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arch 1956 VOL. 15 - No.l =» PARTI

PUBLISHED IN TWO PARTS

LUET UILO
Ls NCCU IN C

CONTENTS

ANNUAL MEETING —-ABSTRACTS OF PAPERS PRESENTED

American Physiological Society. . , ; ]
American Society of Biological Chemists. . . : 208

American Society for Pharmacology and Experimental

Therapeutics. .... 393
American Society for Experimental Pathology. . a 505
American Institute of Nutrition... - es 541
American Association of Immunologists. . 580
Indexes i

Published quarterly by the
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arch 1956 FEDERATION PROCEEDINGS i

Federation Proceedings

Published by

FEDERATION of AMERICAN SOCIETIES
for EXPERIMENTAL BIOLOGY

American Physiological Society

American Society of Biological Chemists

American Society for Pharmacology and Experimental Therapeutics
American Society for Experimental Pathology

American Institute of Nutrition

American Association of Immunologists

EDITORIAL BOARD FEDERATION PROCEEDINGS is published quarterly.

The March issue consists of the Abstracts (Part I) of the

. C. Burton apers presented at the scientific sessions, and the Program
caneeeaee (Part i) of the Annual Meeting of the Federation. The
F. S. CHEEVER July and September issues contain symposia and other
special papers presented at Federation meetings as se-

R. W. EnceL lected by the Editorial Board. The December issue con-

<a tains the membership list and other matters pertinent to
Sa ‘i the Constituent Societies of the Federation.

Puitip HANDLER
Entered as second class matter March 16, 1942, at the Post

Car C, PFEIFFER Office at Washington, D. C. under the Act of March 38,

1879. Copyright 1956, by the Federation of American
Mitton O. Lee Societies for Experimental Biology. Printed at Waverly
Managing Editor Press, Baltimore. Md. All rights reserved.

SUBSCRIPTION PRICE: $7.50 per year ($8.00 Canada, $8.50 foreign) payable in advance.
Single Issues: March, Part I (Abstracts) $5.00, Part II (Program) $1.00; July and September,
$2.00 each; December, $3.00. Subscriptions and orders should be sent to:

Federation Proceedings
9650 Wisconsin Avenue Washington 14, D. C.

it
ci FEDERATION PROCEEDINGS Volume 15 ie |

ne 16

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me 16

An experiment in raising

rats free of certain pathogens.

C. N. Wentworth Cumming and Cloyd Elias

Carworth Farms, Inc., New City, Rockland County, New York

The most prevalent natural infection in rats
today is caused by disease-producing agents with
an affinity for the respiratory tract. These disease-
producing agents have been isolated by Nelson!
of the Rockefeller Institute for Medical Research.
They are a pleuropneumonia-like organism and a
virus and they can occur singly or together in the
same animal. In order to carry on his investigative
work, Nelson has for some years maintained a
colony free of these agents, the original breeding
stock of which was obtained from Reyniers of the
Lobund Laboratories at Notre Dame.

For years many research workers have com-
plained of the experimental complications result-
ing from using rats with respiratory infections.
Much time and money has been wasted on obtain-
ing equivocal results or, in some cases, no results
at all. In an attempt to correct this situation we
decided to attempt production of rats free from
the agents which produce respiratory disease.
Pregnant rats were obtained from Nelson’s res-
piratory-disease-free colony in 1953. Concurrently
several litters were obtained from our CF (Wistar)
rats by Caesarean operation and transferred asep-
tically to the Nelson rats post partum, the latter
having been held in isolation. This transfer was
successfully accomplished in the case of two litters,
one mother raising seven young and the other two.
At this point all Nelson rats were sacrificed except
the two megthers which had reared young. These
were sent to Nelson’s laboratory for examination
and found to be free of all respiratory infections.

The nine animals mentioned above became the

1 §tudies on Endemic Pneumonia of the Albino
Rat. IV. Development of a Rat Colony Free from
Respiratory Infections, Journal of Experimental
Medicine, 1951, 94: 377.

vi FEDERATION PROCEEDINGS

foundation stock for our present CFN colony of
rats. This colony has been kept in isolation and
earéfully observed. All older animals discarded
from the colony have been tested for the presence
of respiratory infections. To date the findings have
all been negative for respiratory disease. In addi-
tion, no animals have been found with supurative
disease of the inner or middle ear, bartonellosis or
salmonellosis.

Rats from this colony have also been sent out
on field trials. When isolated from infected rats,
even though kept under adverse environmental
conditions of temperature and humidity, the test
rats have persisted free from the above mentioned
infections for a period of 1 year. Others which were
placed in the same room with infected rats have
remained free from these complications for 9
months. After 12 months of exposure, the highest
incidence of bacteriologically positive animals was
50% and none of these showed any clinical symp-
toms.

In 1955, because of the satisfactory results, it
was decided to construct a special building to
house this CFN colony. This building incorporates
all the safeguards deemed practical to maintain
the colony free from the specific pathogens which
have been eliminated. A description of this build-
ing has been published in the Carworth Farms
Quarterly Letters?. The first animals were trans-
ferred to their new quarters in December 1955 and
it is anticipated that a few animals will be avail-
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tween 1,200 and 1,500 rats per week is expected to
be reached in September, 1956.

2Carworth Farms Quarterly Letter No. 40,
January 1, 1956.

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viii FEDERATION

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TH
PROCEEDINGS Volume 16 #arch

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k-

me 16

Progressive brain damage
is most frequently associated
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often resulting from a variety of causes.

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Syndrome. Although the primary disease is
very different in these various entities, the
final pathological findings are remarkably
similar, capillary fragility.

March 1956

the Little Stroke

Whenever the ophthalmoscopic examination
reveals hemorrhagic areas, it is reasonable to
assume a comparable process is going on in

the brain.

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manifested by: reduced intellectual activity,
mental confusion, impaired judgment,
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appetite, weakness and early fatigability.

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fragility have always been a difficult problem.
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CTS OF QORIGINAL RESEARCH

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ALESSI, Cb, Hci

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Experimental Biology

There is no provision in the Federation Constitution for individual membership. An indi-
vidual wishing to associate himself with the Federation must become a member of one or
more of the six constituent Societies; such membership entitles him to present papers at sis
the annual meetings of the Federation and to receive the quarterly publication,
FEDERATION PROCEEDINGS. Since requirements and procedures for election to
membership vary among the six Societies, the following information is provided: ‘

1. American Physiological Society. Any resident of North America who has conducted
and published meritorious original researches in physiology shall be eligible for member-
ship. Names of candidates must be proposed in writing by at least two members of the
Society, and from these names the Council shall nominate candidates for election to mem-
bership at a regular meeting of the Society. For further information and proposal forms,
address Dr. M. O. Lez, Executive Secretary, 9650 Wisconsin Ave., Washington 14, D.C.

2. American Society of Biological Chemists. Qualified investigators who have conducted
and published meritorious original investigations in biological chemistry are eligible for
membership. Nominations shall be made and seconded by members of the Society and shall
be submitted to the Council, who shall determine eligibility and make recommendations to
the Society at any regular meeting. Nomination blanks and further information may be
obtained from Dr. Pxttip HANDLER, Secretary, Duke University School of Medicine, Dur-
ham, N. C.

3. American Society for Pharmacology and Experimental Therapeutics. Any person who
has conducted and published meritorious investigations in pharmacology or experimental

therapeutics and who is an active investigator in one of those fields, shall be eligible to :
membership. Candidates shall be proposed by two members who are not members of the A
Society Council, for consideration and recommendation by the Membership Committee R
and the Council. Further information and application forms may be obtained from Dr. C. C. if

PreirFeR, Secretary, Emory University College of Medicine, Emory University Ga.

4. American Society for Experimental Pathology. Any American investigator who has
contributed meritorious work in pathology within three years prior to his candidacy is
eligible for membership. Candidates shall be nominated by two members and the nomina-
tions approved by the Council for election by Society members at a regular meeting.
| Additional information and nomination forms may be obtained from Dr. C. C. Erickson,
University of Tennessee, Institute of Pathology, Memphis 3, Tenn.

5. American Institute of Nutrition. Qualified investigators who have independently con-
ducted and published meritorious original investigations in some phase of the chemistry
or physiology of nutrition, and who have shown a professional interest for at least five
years, are eligible for membership. Nominations shall be made and seconded by members
of the Society and shall be submitted to the Council for recommendation to the Society at
| any regular meeting. For nomination blanks and additional information address Dr. R. W.

ENGEL, Secretary, Virginia Polytechnic Institute, Blacksburg, Va.

6. American Association of Immunologists. Any qualified person engaged in the study of
problems related to the purpose of the Association may apply for membership. Candidates
must be nominated by two members of the Association and applications must be accom-
panied by letters of recommendation, curriculum vitae and a list of reprints of publications.
The Council shall determine eligibility and submit the names of eligible candidates to the
membership at the annual meeting. For further information and application blanks, address
Dr. F.S. CHEEvVER, Secretary, University of Pittsburgh, Graduate School of Public Health
Pittsburgh, Pa.

xvi FEDERATION PROCEEDINGS Volume 16

Mar

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March 1956 FEDERATION PROCEEDINGS xvii

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XViii FEDERATION PROCEEDINGS Volume 16

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THE AMERICAN

PHYSIOLOGICAL

SOCIETY

Seventy-sixth Meeting

Atiantic City, New Jersey, Aprit 16-20, 1956

An asterisk * following an author’s name indicates “by invitation”

|. Effect of epinephrine and nor-epinephrine
on thyroidal release of thyroid hormones in
dogs. NorMAN B. ACKERMAN AND WALTER L.
Arons (introduced by J. E. Ruoaps). Harrison
Dept. of Surgical Research, School of Medicine,
Univ. of Pennsylvania, and the Dept. of Medicine,
Hosp. of the Univ. of Pennsylvania, Philadelphia.
The effect of epinephrine and nor-epinephrine
on the release of thyroid hormones from the thy-
roid gland of dogs has been studied. A large tracer
dose of I! (100-500 uc) is first administered to the
experimental animal. Forty-eight to 72 hr. later
the dog is anaesthetized and serial determinations
of the radioactive protein bound iodine (PBI'*)
are carried out on blood from a cannulated thyroid
vein. These levels were followed during a 2-hr.
control period and during a 2-hr. experimental in-
fusion period of epinephrine or nor-epinephrine.
In all 7 dogs studied during the epinephrine in-
fusion a definite rise in PBI‘! occurred within
15-100 min. after the start of the infusion. In 2
animals studied following nor-epinephrine admin-
istration a similar increase in PBI'* in the thyroid
vein was noted. Radio-paper chromatographic
studies indicated that the rise in PBI'*' was re-
lated to increases in both radioactive thyroxine
and triiodothyronine. Control experiments re-
vealed no change in the thyroid vein PBI'*! over
a4-hr. period. A comparison of the changes in the
PBI?*! of the thyroid vein noted after epinpehrine
and nor-epinephrine infusion with those following
similarly administered thyroid stimulating hor-
mone will be presented.

2. Sodium and potassium alterations in red
cells of patients with sicklemia. JoHN Q.
Apams (introduced by 8. R. Brugscn). Div. of
Obstetrics and Gynecology, Univ. of Tennessee
College of Medicine, Memphis.

Previous reports have (J. Clin. Investigation 31:
406, 1952) introduced the idea that red cells con-
taining a high proportion of hemoglobin S lose K
and gain Na upon sickling. The mechanism by
which such a transfer takes place is not clear and

it is possible that no transfer of ions occurs. This
may be a deceptive phenomenon produced by one’s
inability to pack the sickled cells tightly and
thereby causing an increased contamination with
plasma. The present study approaches the prob-
lem by determinations of the relative concentra-
tions of hemoglobin in the non-oxygenated and
oxygenated samples and a comparison of these
with the concentrations of K and Na found in the
cells and plasma of each sample. By comparing
these ratios some interesting facts regarding the
so-called transfer of ions in sickle cell anemia be-
come apparent. The hemoglobin, K and Na con-
centrations have been determined in 13 normal,
17 sickle cell trait, and 10 sickle cell anemia blood
samples. The decrease in K and hemoglobin, and
the increase in Na in the non-oxygenated samples
of sickle cell anemia blood are significant when
compared with the normal and trait bloods. How-
ever, there is no significant difference between the
decrease of hemoglobin and the decrease of K in
the sickled cells. The degree of change of K and Na
in the sickled sample is almost exactly what would
be expected with the observed decreases in hemo-
globin. This indicates that these changes are due
to dilution and contamination of the cells with
plasma.

3. Ionic and hemodynamic alterations in
hyperthermic dogs. HELMER P. AGERSBORG,
JR.,* GEoRGE BARLOW* AND RicHarp R. Over-
MAN. Clin. Physiology Labs., Univ. of Tennessee
College of Medicine, Memphis.

A mixture of T-1824 and Na*‘, K*?, or P®? was
administered intravenously to dogs. This was fol-
lowed by rapid serial arterial sampling. Subse-
quent analysis allowed measurement of plasma
volume, whole blood volume, cardiac output,
peripheral resistance and the rates of disappear-
ance of isotope from the vascular system. Inde-
pendent determinations gave concomitant meas-
urement of blood pressure, heart rate, hematocrit
and electrocardiogram. The dogs were then sub-
mitted to a controlled ambient temperature

2 FEDERATION

capable of elevating the rectal temperature to
42.5°C. This temperature was maintained for 1
hr. and the variables listed above remeasured.
Increments of high significance over control ob-
servations were noted with regard to Na disap-
pearance rate, heart rate, respiratory rate, he-
matocrit, plasma protein concentration and
plasma and cell potassium concentrations. Analy-
sis of the data obtained concerning cardiac output,
plasma volume and blood pressure revealed de-
creases of high significance. No change was shown
to exist in isotope ‘spaces,’ blood volumes, and K
and P rates of disappearance. The alterations in
electrocardiograms exhibit a strong similarity to
those occurring in hyperpotassemia. Possible
mechanisms of the changes and interrelationships
of the variables in the hyperthermic state will be
discussed. (Supported by a Grant-in-Aid from
the P.H.S.)

4. Passage of colloidal particles through
dermal capillaries under influence of his-
tamine. JoHNn F. ALKSNE* AND H. STANLEY
BENNETT. Dept. of Anatomy, Univ. of Washing-
ton, Seattle.

Janesé in 1947 and the Matoltsys in 1951 demon-
strated that dermal capillary endothelial cells of
rats and mice under the influence of histamine
solution applied topically to the overlying epi-
dermis would phagocytize India ink particles
made available by intravenous injection. After
24-48 hr. the carbon particles were found in nearby
histiocytes. We have performed similar experi-
ments on mice, using a colloidal suspension of
mercuric sulfide particles instead of carbon. The
mercuric sulfide particles range from 30-250 A in
diameter, and are hence within the size range of
plasma protein molecules. These particles were
found to penetrate completely and promptly the
endothelium of dermal capillaries under the in-
fluence of histamine. Twenty minutes after an
intravenous injection of the particles they could
be detected easily with the light microscope in
extravascular macrophages underlying areas of
epidermis painted with a 2% solution of histamine
hydrochloride; 45 min. after injection larger ac-
cumulations of particles were observed in the
macrophages. Mercuric sulfide particles were also
seen within the cytoplasm of endothelial cells of
large and small capillaries, arterioles and venules.
Very few particles were seen in endothelial cells
or in macrophages in the dermis underlying areas
to which no histamine had been applied. The mer-
curiec sulfide particles in endothelial cells of
histamine treated areas are thought to be in pas-
sage to extravascular fluid compartments. (Sup-
ported in part by grant from Life Insurance
Medical Research Fund.)

PROCEEDINGS Volume 16
5. Oxygen consumption and lactate produe-
tion of rat diaphragms and liver slices be-
fore, during and following severe hypoxia,
Norman R. AuPERT, JOSEPH R. Davis Anp
Ropney W. ENGLAND (introduced by RayMonp
C. IncranaM). Dept. of Physiology, Univ. of

Illinois College of Medicine, Chicago.

Following a period of hypoxia the oxygen con-
sumption in unanesthetized dogs gradually re-
turns from depressed levels to control levels
without any evidence of an excess consumption,
At the same time lactate is produced during the
hypoxia and then removed during the recovery
(Am. J. Physiol. 179: 614, 1954). The following
experiments were designed to ascertain the role
which individual tissues play in this picture of re-
covery from hypoxic stress. Tissue oxygen con-
sumption was measured before, during and follow-
ing a 60-min. period of hypoxia by means of the
Warburg manometric apparatus. During each of
these periods lactate production and removal was
evaluated. In the diaphragm the oxygen consump-
tion was depressed from a control value of .106
ul/mg/min. (.19-.08) to .01 ul/mg/min. (.08-.00).
During the recovery the oxygen consumption
never was greater than the control values. The
lactate produced was independent of the amount
of oxygen missed during the hypoxia. The oxygen
missed ranged from .5 to 8.5 wl/mg whereas the
lactate produced was essentially the same for
each of the tissues (1.6 ug/mg). Furthermore for
the first 30 min. of recovery lactate was produced
at the same rate as during the hypoxia. In the
liver slices the oxygen consumption was depressed
from a control value of .20 wl/mg/min. (.26-.15)
to .02 ul/mg/min. (.06-.00). During the first 10
min. of recovery the oxygen consumption was
greater than the control values. This excess, how-
ever, was only about 10% of the oxygen missed.
The oxygen missed ranged from 5.5 ul/mg to 12
ul/mg. The lactate produced, 3.77 ug/mg (1.86-
10.49), was not dependent on the oxygen missed.

6. Local response in an excitable membrane.
Mario ALTAMIRANO. Dept. of Neurology, College
of Physicians and Surgeons Columbia Univ., New
York City.

The normal response of the innervated mem-
brane of the electroplaque is an all-or-nothing
propagated spike. Local responses, i.e. non-
propagated activity whose amplitude is propor-
tional to the stimulus strength, may also be
elicited. However, this activity may be found only
after 1) eserine, procaine, d-tubocurarine, excess
or lack of potassium, lack of sodium, damage of
the membrane, etc., and 2) when just threshold
stimulation is used. In group 1 the excitability is
always decreased; spikes of normal magnitude are
unable to stimulate adjoining regions. The simul-
taneous recording of the transmembrane potential

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March 1956

at varying distances of a pin-point stimulating
electrode shows that the magnitude of the local
response is proportional to the area excited (i.e.
to the total current delivered by the responding
membrane). However, if the whole membrane is
simultaneously excited, only all or nothing re-
sponses are observed. This finding shows that
even under the conditions classified under / the
membrane discharges by an all-or-nothing change.
The local activity is not the result of an intrinsic
quality of the membrane, but depends upon the
stimulating and recording arrangement. A thresh-
old stimulus often excites only discrete regions
that have either transitorily or permanently lower
thresholds than the rest of the membrane. The
current delivered by the small active areas is not
sufficient to produce a threshold voltage drop
across the adjoining membrane; the response does
not propagate. The membrane of the stimulated
area probably undergoes similar changes as in an
all or nothing response. In conclusion, there is not
evidence that a membrane response exists in the
electropiax basically different from that resulting
in an all-or-nothing spike.

7. Effects of high altitude exposures on dogs
and on their susceptibility to endocarditis.
Paut D. ALTLAND AND BENJAMIN HIGHMAN.
Natl. Inst. of Arthritis and Metabolic Diseases,
Natl. Insts. of Health, Bethesda, Md.

Studies were made on 15 dogs exposed to simu-
lated altitudes of 25,000 ft. 6 hr. daily 5 times
weekly for 1-27 months. Hematologic studies will
be reported. Pathologic studies revealed marked
vascular engorgement. Changes in cardiac valves
and other lesions attributable to high altitude
were similar but less frequent and severe than in
similarly exposed rats (Arch. Path. 48: 503, 1949).
Nine dogs with hematocrits from 67 to 81 (exposed
5-8 months) received bacteria Usually nine 2-cc
injections of 5-hr. broth cultures were given intra-
venously within 2 wk. Four dogs received Staphylo-
coccus aureus. One dog, which died 8 days after the
first of 5 injections, showed mitral and aortic bac-
terial vegetations. Another, dying in 3 days,
showed a nonbacterial valvulitis. Both showed
renal infarcts, and widespread suppurative and
hemorrhagic lesions. Two dogs, killed in 14 and
20 days showed no cardiac changes attributable to
bacteria. Five altitude dogs received Streptococcus
mitis. One dog apparently unaffected was not
killed. One dog was killed in 14 days, and 3 died
in 4, 6 and 8 days; all showed a nonbacterial
valvulitis and 1 showed renal infarction with bac-
teria No significant inflammatory lesions were
observed in 10 ground level controls given bacterial
injections and in 5 uninjected altitude controls.
A 6th dog is alive after continuing altitude ex-
posures for 27 months. These findings suggest that

AMERICAN PHYSIOLOGICAL SOCIETY 3

susceptibility to endocarditis is moderately in-
creased in dogs exposed to simulated high alti-
tudes.

8. Effect of cortisone on carbohydrate me-
tabolism as studied with C glucose in
hypophysectomized and adrenalectomized
dogs. N. ALTSZULER, R. STEELE, J. S. WALL
AND R. C. vE Bopo (introduced by S. J. Far-
BER). Dept. of Pharmacology, New York Univ.
College of Medicine, New York City, and Biology
Dept., Brookhaven Natl. Lab., Upton, N. Y.

A method employing uniformly labeled C*
glucose (STEELE ef al., Federation Proc. 14: 286,
1955) was used to determine the size and the
inflow-outflow rate of the glucose pool in trained,
unanesthetized dogs. The glucose pool in hy-
pophysectomized dogs, 0.198+0.018 gm/kg, was
smaller than in normal dogs, 0.284 + 0.011 gm/kg,
and the rate of glucose inflow into the plasma,
2.59 + 0.26 gm/m2/hr., was also less than in nor-
mal animals, 3.80+0.19 gm/m?/hr. During these
experiments the plasma glucose level remained
unchanged, therefore the glucose outflow rate
equalled the glucose inflow rate; this indicates a
lower glucose uptake by the tissues. The latter
suggests an adaptation to the limited glucose in-
flow: On a cortisone regimen (0.8-1.5 mg/kg/day
for 8-16 days) the glucose pool in the hypophysec-
tomized dog was increased and the glucose inflow-
outflow rate was restored to normal. The increased
outflow may reflect on increased insulin secretion
in response to the improved inflow of glucose into
the plasma. In adrenalectomized dogs, neither the
glucose pool nor the glucose inflow-outflow rate
was as low as in the hypophysectomized animals.
The implications of these findings will be dis-
cussed.

9. Is the cuneate nucleus a simple mono-
synaptic relay? VAHE E. AmassiAn. Dept. of
Neuropsychiatry, USAF School of Aviation Medi-
cine, Randolph Air Force Base, Texas.

Single unit analysis of activity in the cuneate
nucleus (Federation Proc. 13: 3, 1954) indicates
that following a shock to the medial lemniscus a
cuneate cell is initially either antidromically in-
vaded, or activated after synaptic delay by the
intranuclear collaterals of cuneate axons described
by Cajal. Such axonal collaterals probably
act during orthodromic excitation because 1)
evoked activity commences late (1-3 msec. ex-
cluding dorsal column relay action) in 1 class of
cuneate cells after arrival at the nucleus of the
peak afferent volley from the radial cutaneous
nerve, despite consistent occurrence of repetitive
discharge (P = 1) at high rates (up to 1550/sec.)
and consistent latency and first interspike inter-
val (variation as low as +20 ysec.). When late
discharge is associated with such powerful excita-

4 FEDERATION PROCEEDINGS

tion, the lag is more likely due to synaptic delay
introduced into the presynaptic pathway than to
long response-time following the arrival of af-
ferent impulses. Early discharge (up to 1 msec.)
in other cells can be converted to late discharge
by weakening the afferent stimulus, but it then
occurs with lower probability, lower repetition
rate and greater temporal variability. 2) When the
dorsal columns are stimulated above C 2 to reduce
temporal dispersion of the afferent volley, cuneate
cells initially discharge electively at times cor-
responding to 1 or 2 or more synaptic delays. Thus,
although some cuneate cells are monosynaptically
excited, others plurisynaptically excited,
through cuneate cells. After-discharge is insig-
nificant after repetitive peripheral stimulation
indicating that little ‘circus’ movement occurs in
cuneate chains.

are

10. Device for electroseparation of proteins.
N. G. AnpERSON. Biology Div., Oak Ridge Natl.
Lab., Oak Ridge, Tenn.

Preparative zone electrophoresis on starch or
agar has generally been limited in capacity, has
used voltages in the kilovolt range, and has re-
quired long periods of time. Heat dissipation was
at right angles to the direction of protein migra-
tion and limits the thickness of the block. Protein
is generally recovered by elution. A new device is
described consisting of a series of plastic frames
supporting a thin sheet (8 mm) of protein in
starch or agar against a thicker (13.5 mm) sheet
of the same. Glass screen, filter paper and cello-
phane hold the supporting material in the frames.
The proteins migrate through the supporting
medium into a narrow space between glass screen
and cellophane, where they are concentrated by
electroconvection and removed continuously or
intermittently from the bottem. Since the break-
through time for a given protein is inversely pro-
portional to its mobility, the emergence pattern,
although shgwing the same peak order as free
electrophoresis, will show small intervals between
fast components and much longer intervals be-
tween slow components. A graphic method for
relating the two patterns is presented. Only 24
volts being used, a soluble liver nucleoprotein has
been isolated from soluble liver proteins in 50
min. Gram quantities of serum albumin have been
separated in a few hours. The volume of the start-
ing layer is 68 ml in the small model described. To
sharpen the starting layer, the current is reversed
to pile up the faster components against the back
cellophane sheet.

ll. Erythrocyte and plasma volumes in the
goat. RuBERTS. ANDERSON AND E. B. Roarrs.*
Biophysics Div., Chemical Corps Med. Labs.,
Army Chemical Center, Md.

Determinations of plasma volumes by I'*! and

Volume 1§

by T-1824 dye and erythrocyte volumes by Cri
have been made. Since many of these have been
simultaneous, the direct as well as the indirect
methods of arriving at the volumes can be com-
pared. Also, as a corollary, the whole body hemato-
crits can be calculated and compared with venoug
hematocrits under different conditions. Measure-
ments have been made in the normal goat before
and after hemorrhage and transfusion of known
blood volumes.

12. Polydipsia evoked by hypothalamic stim.
ulation in the goat (motion picture). B.
ANDERSSON* AND S. M. McCann. Dept. of
Physiology, Kungl. Veterindérhégskolan, Stock-
holm, Sweden.

The polydipsic effect of injections of hypertonic
saline solution into the hypothalamus of goats has
been described (Experientia, VIII/4: 157, 1952).
In the present study, hypothalamic stimulation
was produced by microinjections of hypertonic
saline and by electrical stimulation, using a modi-
fication of the technique of Hess. The drinking re-
sponse of the goats was filmed. Microinjections of
less than 0.01 ml of 3% NaCl solution induced
drinking of large volumes of water when performed
within a fairly wide portion of the middle hypo-
thalamus. Electrical stimulations of the same
region where osmotic stimuli were effective gave
a more reproducible polydipsic effect. An animal
weighing about 40 kg could drink more than 151.
of water during the course of an experiment, and
the stimulatory effect could be repeated at will.
The latency before the onset of drinking varied be-
tween 6 and 45 sec.; drinking usually continued
without interruption throughout stimulation and
for 3-6 sec. afterward. The parameters of stimula-
tion were 0.5-1.5 v. and 50 c/s. In additional ex-
periments, not on film, lesions were made in the
hypothalamus of dogs. Destruction of a substan-
tial portion of the same region of the hypothal-
amus where stimulation produced polydipsia in
the goat resulted in a partial or complete loss of
thirst in the dog, although a partial recovery of
drinking occurred in most cases.

13. Carbohydrate metabolism of skeletal
muscle in man during the night. REvuBIN
ANDRES AND KENNETH L. Z1ERLER. Depts. of
Environmental Medicine and Medicine, Johns
Hopkins Univ. and Hosp. Baltimore, Md.
Certain metabolic events occurring in resting

skeletal muscle (the forearm) were studied inter-

mittently over the hours 10 p.m.—11 a.m. by meas-
uring appropriate arterio-venous concentration
differences and forearm blood flow in 3 normal
men. No food was taken after 6 p.m. Blood flow,

Os consumption and COs: production showed no

pattern over these hours. Lactate was continu-

ously produced but was also patternless. Arterial

Mar

cone
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March 1956

concentrations of these 3 metabolites were con-
stant. In contrast, arterial glucose concentration
fell progressively in all 3 subjects and was ac-
companied by progressive decreases in glucose
A-V difference and glucose uptake. Lactate pro-
duction may be considered a measure of the
fraction of glucose being anaerobically metab-
olized; the remaining fraction of glucose may then
be considered available for oxidation, and the
contribution of glucose as oxidative substrate
thus assessed. Lactate production initially ac-
counted for only about } of the glucose uptake,
but as glucose uptake fell, it finally accounted for
all. The fraction of O2 uptake spent in oxidation
of glucose fell progressively. Even in the early
hours glucose uptake was insufficient to account
for all the O2 consumption and towards the end of
the experimental period accounted for less than }
the O» uptake in all subjects. There was no con-
sistent trend in forearm R.Q.; the over-all mean
R.Q. of 0.82 supports the conclusion that carbo-
hydrate is not the major metabolic substrate
during the nocturnal half of the day. (Supported
grants from ONR, NIH and Muscular Dystrophy
Associations. )

l4. Brain acetylcholine and behavior. M. H.
APRISON AND P. NaTHANn.* Galesburg State Re-
search Hosp., Galesburg, Ill.

When DFP is injected into the right common
carotid artery of rabbits, most animals exhibit
compulsive turning movements to the left (‘left-
ers’), some show no change in their normal mode
of progression, and a few turn toward the right
(‘righters’). The marked asymmetry in the reduc-
tion of cholinesterase activity in areas of the right
and left cerebral cortex and caudate nucleus in a
given rabbit was correlated with its behavior.
Stimulation of the cortex was postulated in an at-
tempt to explain the mechanism of compulsive
circling. It was suggested that accumulated
acetylcholine was the agent producing this phe-
nomenon. Free acetylcholine has now been deter-
mined in the right and left cortex and caudate
nucleus of normals, ‘lefters’ and ‘righters.’ In
normals, the acetylcholine concentration was the
same on both sides of the brain, the caudate
nucleus being approximately 7 times higher than
the cortex. In ‘lefters’ marked asymmetry between
the 2 sides of the brain was noted. The acetyl-
choline content of the right cortex and caudate
nucleus were approximately 3 times higher than
the corresponding left brain areas. Finding the in-
creased acetylcholine content in the right cortex
substantiates the earlier hypotheses for ‘lefters.’
In ‘righters’ it was found that the acetylcholine
concentration of the left cortex was higher than
the right. In the case of the caudates in this series
the reverse was true. Therefore, the exact role of

AMERICAN PHYSIOLOGICAL SOCIETY 5

the caudate nucleus in our preparation is not
known at present. However, the biochemical evi-
dence suggests that stimulation on one side of
the cerebral cortex causes compulsive circling
toward the contralateral side.

15. Changes in cardiovascular system fol-
lowing total body x-irradiation. W. D.
ARMSTRONG AND W. O. CasteR.* Dept. of
Physiological Chemistry, Univ. of Minnesota,
Minneapolis.

Simultaneous studies of chemical and physio-
logical alterations in the cardiovascular system of
the 350-gm male white rat have been made both
under normal conditions and following the ad-
ministration of 700 r (an LD dose) of 140 kv
x-irradiation. Many of the observed changes
occurred either during the first day or two, or at
the 6-8 day period, i.e. at the start of either the
acute or LD death periods. Among the statisti-
cally significant changes following irradiation are
the following: 1) an increase in venous pressure at
12 hr. and again in 5-12 days, 2) a decrease in
heart potassium concentration at 3 and 8 days,
8) a decrease in heart DNA concentration at 2-3
and 8 days, 4) a progressive increase in the relative
length and amplitude of the first heart sound
which becomes significant by 10 days, 5) an ir-
regularity in the heart beat as shown by a random
variation in the R-height at 10 days, 6) an increase
in the height of the T-wave as measured from the
8’-notch (the T-wave is normally fused with the
declining limb of the R-wave so that the T-wave
starts approximately when the R-wave has re-
turned only half-way to the isoelectric point; this
change largely represents a downward movement
of the S’-notch rather than an increase in the
T-wave above the isoelectric point), and 7) a
significant increase in plasma volume (excluding
the chambers) and extracellular space in the heart
from 6-10 days following x-irradiation.

16. Nervous influences on pulmonary circula-
tion. Dominco M. Avrapo, Jr., ALBERT H.
NipEN* AND Cart F. Scumipt. Lab. of Pharma-
cology, Univ. of Pennsylvania School of Medicine,
Philadelphia.

The left lower lobe of an anesthetized dog was
vascularly isolated and perfused (via its lobar
artery) at a constant flow with venous blood from
a donor dog. The additional insertion of broncho-
spirometer tubes allowed administration of gas
mixtures independently to either the left lower
lobe or to the other (nonperfused) lobes. In 30
such cross-circulation experiments, a rise in per-
fusion arterial pressure, indicating vasoconstric-
tion of the perfused lobe, was brought about by
each of the following procedures: increased intra-
cranial pressure, inhalation of 5-10% Oz, inhala-

6 FEDERATION PROCEEDINGS

tion of 5-15% COs: in air, reduction of carotid
sinus pressure by occlusion of both common
carotids, intravenous injection of veratridine
(Bezold-Jarisch reflex) and intravenous injection
of glass beads (60-240 »). The pulmonary vaso-
constriction response was eliminated by excision
of T,-T, sympathetic ganglia. While pulmonary
embolization elicited an interlobar vasoconstrictor
reflex no interlobar reflex was activated by atelec-
tasis, hyperinflation and administration of the
above gas mixtures. All the above procedures
establish the existence of nervous influences upon
the lung vessels but do not indicate the exact site
of vasoconstriction, e.g. precapillary, postcapil-
lary or pulmonary arteriovenous communications.
Until this is known, the functional significance of
pulmonary vasoconstrictor nervous influences
will remain obscure. This neurogenic response
should be considered with other factors known to
regulate the pulmonary circulation. In the intact
dog (without lung perfusion), the sympathetic
vasoconstrictor reflexes occur simultaneously
with, and are probably overshadowed by, altera-
tions in pulmonary blood fiow and local changes
in vascular resistance.

17. Production of a permanent state of excita-
tion in mice and rats with §8-iminodipro-
prionitrile. H. Azima anp B. Grap (introduced
by R. A. CLeEGHORN). McGill Univ. and Allan
Memorial Inst., Montreal, Canada.

The subcutaneous daily injection of 4 mg/kg of
88-Iminodiproprionitrile in mice produced a state
of motor excitation and agitation from the 3rd
day on, animals being very active and running
around persistently. This state of excitation had
the following characteristics: 1) It was perma-
nent, being observed even 1} yr. after treatment
stopped; 2) it was accompanied by a relative state
of hypotonia, motor incoordination and groping
movements of the head; 3) it did not interfere
with normal activities such as feeding and sleep-
ing; 4) it was associated with a state of over-
alertness, animals being over-responsive to
stimuli; 5) it could be controlled by the subcutane-
ous administration of reserpine, as long as the
drug was used. In rats, in addition to the above
observations, the following effects were noted:
animals showed a combination of over-alertness
and somnolence; they manifested a marked startle
reaction to stimuli, but would become somnolent
if left alone. They also showed a decrease in their
epileptogenic threshold.

18. Mechanism of participation of the system
ascorbic acid — dehydroascorbic acid in
hepatic corticosteroid metabolism. HABEEB
Baccnus AND ALBERT F. Depons. Dept. of
Physiology, George Washington Univ. School of
Medicine, Washington, D. C.

Volume 15

The influence of ascorbate on the in vitro hepatie
metabolism of corticosteroid hormones has been
demonstrated (Endocrinology 53: 617, 1953; 57;
531, 1955). Recent work suggests that the mech.
anism of action of ascorbate involves the oxida-
tion-reduction potencies of the system ascorbic
acid — dehydroascorbic acid. Ascorbic acid added
to the phosphate buffer system (px 7.40) rapidly
becomes oxidized to dehydroascorbic acid. In the
presence of surviving liver tissue the oxidation to
dehydroascorbic acid is inhibited. In the presence
of liver tissue the A*,3-ketone structure of cortico-
steroids is reduced; when the system (ascorbic
acid — dehydroascorbie acid) is added to such 4
preparation, the reduction of the A‘,3-ketone
structure is inhibited. Ascorbic acid, or dehydro-
ascorbic acid, fails to directly alter the A‘,3-ketone
structure. The data suggest that the system
ascorbic acid — dehydroascorbic acid provides a
competitive hydrogen acceptor system which
preferentially becomes reduced by the liver, in
such a manner that the reduction of A‘,3-ketones
is depressed. Complete studies on the levels of
ascorbic acid, dehydroascorbic acid, diketo-
gulonic acid, as well as the corticosteroids and
their metabolites in the in vitro system will be
presented. (Aided by contract with the Office of
Naval Research, and by a grant (A-852) from the
Natl. Inst. of Arthritis and Metabolic Diseases.)

19. Specific cellular sites of ferritin oxida-
tion-reduction in the liver. Sitvio Bagz,
EpHrAIM SuHorr,! S. G. Srrkantia,* Iris
ForsBEes* AND ANNE CARLETON.* Dept. of Medi-
cine, Cornell Univ. Med. College, New York City.
It has now been found possible to assign to

specific cellular components of the liver specific

reactions in the intermediary metabolism of
ferritin within that organ. Utilizing a procedure
described by Rous in 1927, consisting essentially
in the preliminary injection of carbonyl] iron fol-
lowed by isolation of the liver cells through a
press and fine silk and their suspension in an
isotonic medium, the reticulo-endothelial (RE)
cells can be separated from the parenchymal cells
by means of a powerful eye magnet in a viable
state suitable for subsequent metabolic studies.
The questions which were explored were whether
the storage of ferritin and its oxidation and redue-
tion are functions of all the liver cells or limited
to specific cell types. The major if not the entire
content of liver ferritin appears to be contained
in the RE cells in a vasoinert form under aerobic
conditions. Exposure of RE and parenchymal
cells to anaerobiosis shows that only the RE cells
release vasoactive reduced ferritin into the
medium and this in small amounts compared to
their intracellular ferritin which is maximally re-

1 Deceased.

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March 1956

duced under,these conditions. On the other hand
only the parenchymal cells can aerobically in-
activate, that is, oxidize vasoactive ferritin
whether in the medium or within the RE cells.
These experiments were carried out with rat liver
and the assays for ferritin made by the rat meso-
appendix test combined with the use of anti-
ferritin serum to achieve specificity.

2. Reduction in parotid amylase after hypo-
physectomy. Burton L. Baker, JEROME H.
KLEGMAN* AND Rospert G. LoGan.* Anatomy
Dept., Univ. of Michigan, Ann Arbor.
Hypophysectomy in the adult rat caused a

striking cytological involution of the acinar cells
in the parotid ‘and reduction in weight of the
gland. The amylase content was determined by
homogenizing the gland, diluting with physio-
logical saline and incubating a portion of the
homogenate with starch. The amount of reducing
sugar released from the starch was quantitated by
the Shafer-Somogyi method and expressed in
terms of maltose by reference to a standard curve.
Seven to 8 days after the operation, the concen-
tration of amylase was equivalent to 2.4 mg
maltose/0.1 mg of parotid as compared with 4.1
mg for the controls. This difference was signifi-
cant at the 0.1% level. The total activity per
gland also was much lower in the hypophysec-
tomized animals. At 39-78 days after the operation
a comparable reduction was observed. Similarly,
when calculated in terms of the total activity per
gland or in relation to body weight, significant
reductions were observed. Thus, the parotid
gland resembles other enzyme-producing cells
associated with the digestive tract in being
regulated by the pituitary gland.

21. Effects of altitude acclimatization on
work capacity. Bruno BALKr, J. G. WELLS
AND JAMES P. Exuts (introduced by Harry F.
ApLER). Dept. of Physiology-Biophysics, USAF
School of Aviation Medicine, Randolph Air Force
Base, Texas.

An attempt was made to measure the reduction
of working capacity during acute and chronic ex-
posure to altitude levels of 14,000 ft. A standard-
ized test of gradually increased work load on a
bicycle ergometer was applied at base level for
controls, in a low pressure chamber and during a
6-wk. stay on Mt. Evans, Colorado. Comparable
physical condition was achieved by a preceding
physical training of 8 wk. duration. In all experi-
ments the oxygen consumption at comparable
work intensities remained practically unchanged.
The pulmonary ventilation (BTPS) was almost
doubled at altitude. Maximal ventilation observed
was 122 1/min. at base level but 170 1/min. after
some acclimatization on Mt. Evans. In the acute
exposure to hypoxia the blood pressure was not

AMERICAN PHYSIOLOGICAL SOCIETY 7

altered. However, with acclimatization to 14,000
ft. preceding systolic and diastolic pressure went
up. The pulse rate exceeded the ground level
values for same work intensities in the range of
light and medium work load, though the pulse
maxima were remarkably (11-14%) lower at alti-
tude than at base level. On the average of 6 sub-
jects the performance tests in the low pressure
chamber showed a reduction in work capacity of
27%. Surprisingly, 6 wk. of acclimatization to an
altitude of 14,000 ft. were not sufficient to raise
this performance level perceptibly. Immediately
after return from the mountain, physical per-
formance was improved above the controls. The
maximal oxygen uptake was increased.

22. Ascites formation without sodium reten-
tion in dogs with thoracic inferior vena
cava constriction and dogs with pulmo-
nary artery constriction. WiuMot C. BALL,
Jr.,* JAMES O. Davis anp M. Jay Goopkinp.*
Natl. Heart Inst., Bethesda, Md.

Seventeen dogs subjected to thoracic inferior
vena cava constriction (IVC) and10 sham-operated
controls were given water ad libitum but no food
for 4 days after surgery. Four additional dogs were
starved for 4 days after constriction of the pulmo-
nary artery (PA). Ten of the IVC dogs and 2 of
the PA dogs formed ascites in amounts up to 840
ec. Inferior vena caval pressure in the IVC dogs
with ascites varied from 186 to 294 mm water; in
those without ascites pressure varied from 160 to
220 mm water. Right atrial pressures in the 2 PA
dogs with ascites were 180 and 170 mm water as
compared with 128 and 90 in those without ascites.
Plasma Na on the last day of starvation averaged
133 mEq/l]. in those dogs which formed ascites
and 143 mEq/l. in those which did not. Mean Na
excretion was 4.6 mEq/day for the IVC dogs and
12.1 mEq/day for the control dogs (P < 0.02);
3.8 mEq/day were excreted by the PA dogs. RPF
was consistently reduced in both IVC and PA
dogs during starvation; GFR was measured in 4
dogs with ascites and showed no decrease from
control levels. The aldosterone activity of urine
from the IVC dogs was markedly greater (P <
0.01) than that of normal dog urine, and sig-
nificantly greater (P < 0.02) than that of urine
from the sham-operated controls.

23. Coronary blood flow and_ regulatory
factors in normal, sympathectomized,
vagectomized and atropinized dogs. T. A.
Ba.ourpas,* J.C. Scotr anp W. B. MacGratu.*
Inst. for Cardiovascular Research and Dept. of
Physiology, Hahnemann Med. College, Phila-
delphia, Pa.

Investigations were made on coronary blood
flow and mechanisms influencing coronary circu-
lation. Observations were made on 32 chest-closed

8 FEDERATION PROCEEDINGS

dogs under morphine and Dial-Urethane-Nem-
butal anesthesia by the nitrous oxide desaturation
method with catheterization of coronary sinus.
Sympathectomy (stellate to T;-Ts) or bilateral
vagectomy was performed at least 6 wk. before
determinations. Atropine 0.1-0.2 mg/kg. was
injected intravenously. The majority of experi-
ments were performed with a mixture of 15% N.O,
64% No, 21% Oc, a few with 20% NO and 80%
O:. Results: average values for normal dogs were:
flow: 77.5 ec/100 gm/min.; coronary vascular
resistance (mean arterial pressure/flow): 1.5,
cardiac index 2.9, left ventricle oxygen consump-
tion: 9.6 cc/100 gm/min., coronary sinus Oz: 4.8
vol. %. The average values for sympathectomized
dogs showed a decrease in flow, L.V. O2 consump-
tion and cardiac index, and an increase in CVR.
Both vagectomized and atropinized dogs had an
increase in flow and L.V. O2 consumption but a
decrease in CVR. A linear relationship between
flow and L.V. O2 consumption and between flow
and coronary resistance was observed in each
group. The findings suggest that the cardiac
sympathetics exert vasodilatory influences on the
coronary vessels. The chronic effects of bilateral
vagectomy or the immediate effects of atropine
suggest a tonic vasoconstrictor function of the
cardiac vagus fibers. (Supported by Grant H-2003
R from the Natl. Heart Inst., PHS, and Hahne-
mann Inst. for Cardiovascular Research.)

24. Chronic, unilateral renal artery catheter-
ization: differential sodium excretion
normal, unanesthetized dogs, and dogs
with congestive heart failure. A. C. BARGER,
A. M. Rupowpnu,* 8S. N. Rokaw* anp F. E.
Yates.* Dept. of Physiology, Harvard Med.
School, Boston, Mass.

A new technique has been developed for the
chronic catheterization of 1 renal artery of the
dog (Rudolph, Rokaw and Barger), with collec-
tion of uriae from each kidney by means of 2
bladder pouches (Desautel). The presence of the
catheter has not altered renal function for a period
up to 6 months. With such a technique it is now
possible for the first time to study the renal re-
sponse to salts and drugs injected directly into
the renal artery of normal, unanesthetized dogs,
and dogs with congestive heart failure. For ex-
ample in the normal dog the slow infusion of
hypertonic saline into 1 renal artery produces a
5-10-fold increase in sodium excretion on the in-
jected side without any change in glomerular
filtration rate or renal plasma flow. In contrast,
in the dog with congestive heart failure, with
normal glomerular filtration rate, but low renal
plasma flow, such an infusion of hypertonic saline
into 1 renal artery produces no increase at all in
sodium excretion on the injected side,—clearcut

Volume if

evidence for increased tubular reabsorption of
sodium in congestive heart failure. The effects of
unilateral injection of cardiac glycosides and
other drugs will also be discussed.

25. Modifications of hemoglobin affinity and
hypoxia tolerance in infant and adult rats
following exposure to low and high O, ten.
sions and irradiation. J. N. BARKER (intro-
duced by F. G. Hau). Dept. of Physiology and
Pharmacology, Duke Univ. School of Medicine,
Durham, N.C.

Infants have hemoglobin with a higher affinity
for O» than that of the adult, and hypoxia toler-
ances are greater in proportion to affinity. High-
affinity hemoglobins (HAHb) and high tolerances
were also found in some adult rats and mice after
exposure to low pO». Further studies indicate that
HAHb appears within 24-72 hr. in all adult rats
exposed for 2 hr. daily to a pO2 of 50 mm Hg,
affinities often exceeding the mean for newborn
rats if exposures are continued for a week. Ex-
posures to a pO» of 27 mm for 10 min. or of 90 mm
for 20 hr. are less effective. Polycythemia does not
necessarily accompany the shift. If HAHb, which
normally occurs only in infants, is a consequence
of intrauterine hypoxia, its production might be
inhibited by high pOs. In newborn rats exposed
to 50% Os for 72-96 hr., the disappearance of
HAHb and of hypoxia tolerance was greatly ac-
celerated, and the infants were more anemic than
controls. Although affinities did not increase on
removal to air, they did on exposure to low pOx,
In collaboration with Dr. A. Ottolenghi, it was
shown that HAHb and greater hypoxia tolerance
also appeared in rats 12+4 days after irradiation
with 600 r, disappeared within 35 days, but re-
appeared on reirradiation. Although radiation
HAHb may result from selective survival of
HAHb-producing cells or some other unknown
mechanism, it might also arise from circulatory
hypoxia of spleen and marrow. Since no HAHb is
found during recovery from severe hemorrhage,
its appearance is probably not related to a specific
phase of erythrocyte development. Splenec-
tomized rats readily produce HAHb on exposure
to low pO. No abnormalities attributable to
presence of HAHb have been observed.

26. Thyroxine analog effects on succinate
and malate oxidation in vitro. S. B. BARKER
AND W. J. Lewis.* Dept. of Pharmacology, Univ.
of Alabama Med. Ctr., Birmingham.

Using a phosphate homogenate of rat heart as

a source of succinoxidase, addition of thyroxine

considerably retards the rapid falling off in ac-

tivity characteristic of this system (GEMMILL,

1952, WiIswELu et al. 1954). These findings have

been confirmed and extended to a variety of

thy
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March 1956

thyroxine -derivatives, analogs and _ isomers.
‘Stimulation’ of succinate oxidation 75-110% as
great as that produced by thyroxine was shown by
many compounds, including 3,5,3’-triiodothyro-
nine (Trit), the thioether analogs of tetraiodo- and
triiodothyronine, tetraiodo- and_ triiodothyro-
acetic acids, tetraiodo- and diiodothyropropionic
acids, tetraiodo- and diiodothyroacrylic acids,
tetraiodophenolphthalein, and N-(3,5-diiodo-4-
hydroxybenzoyl)-3,5-diiodotyrosine (B-5522). It
should be noted that Trit is about 4 times as active
in vivo as thyroxine, while the last 2 are inactive,
with the remainder ranging from 10 to 100% as
active as thyroxine. Thus, the in vitro enhance-
ment of succinoxidase activity is different from
the in vivo effects of thyroxine, at least on succin-
oxidase, and probably on metabolism in general.
Paralleling these succinoxidase ‘stimulations’ for
the most part are depressions of malate oxidation,
thus confirming the concept of Clarke and Ball
(1955) that the thyroxine succinoxidase effect is
operating by preventing accumulation of oxalo-
acetate. The thyroxine, Trit and B-5522 effects
are also present with phosphate homogenates of
diaphragm and liver, but are essentially the same
for comparable tissues from euthyroid and hypo-
thyroid animals. (Supported by grants from the
Smith, Kline and French Fndn. and the American
Cancer Society.)

27. Blood levels of 17-hydroxycorticosteroids
in hypothermic dogs. GroRGE BaR.ow,*
HetMER P. AGERSBORG, JR.* AND RicHarD R.
OveRMAN. Clin. Physiology Labs., Univ. of
Tennessee College of Medicine, Memphis.
Utilizing the Nelson-Samuels method for de-

termining 17-hydroxycorticosteroids, measure-

ments of plasma steroid levels have been made on

13 anesthetized dogs subjected to exogenous

hyperthermia induced in a hypertherm cabinet.

After obtaining control values for mean blood

pressure, rectal temperature, plasma and red

blood cell electrolytes, and plasma steroid levels,
the dogs were exposed to an ambient temperature
of 50°C for periods sufficient to provoke a rise in
rectal temperature to 42.5°C, which was then
maintained for 1 hr. The above-mentioned vari-
ables were remeasured at the following intervals:
when the rectal temperature reached 42.5°C 3 hr.
and 1 hr. after the rectal temperature reached
42.5°C, and again as the rectal temperature re-
turned to normal or before the dogs succumbed.

The animals evidenced an average change in

plasma steroids of +340% after their rectal

temperatures had been maintained at 42.5°C for

1 hr. This increment was steadily progressive

throughout the warm-up phase and was main-

tained, even magnified in some instances, after the
dogs were permitted to cool. Concomitant with

AMERICAN PHYSIOLOGICAL SOCIETY 9

plasma steroid increases were elevations in
plasma K concentration averaging 3 mEq/I.
above normal. It is suggested that the stress of
hyperthermia, which proved fatal within 1-24 hr.
after the animals were removed from the cabinet,
augmented by hyperpotassemia causes the release
of large amounts of adrenal cortical steroids.
(Supported by grant-in-aid from the Public
Health Service.)

28. Cytochemistry of filamentous Escherichia
coli. Ropert C. BARNETT AND GRAcE Morrow
(introduced by E. L. Porter). Med. Physics
and Carter Physiological Lab., Univ. of Texas
Med. Branch, Galveston.

Localization of succinic dehydrogenase by neo-
tetrazolium reduction has been compared in
normal and penicillin produced filamentous
E. coli B. Presence of the enzyme was indicated by
stainable granules which appeared in the presence
of succinate but did not appear in the absence of
succinate nor in the presence of succinate plus
malonate. When succinate is added, darkly stained
granules appear at both tips of normal E. coli;
whereas, the stainable granules are separated at
two cell lengths in the filamentous forms. Pre-
incubation for 3 hr. with compounds that have
been shown to fragment the filamentous forms
(cysteine, glutathione, Krebs cycle intermediates
or adenine containing compounds) stimulate the
appearance of additional granules spaced at one
cell length along the filaments. Similar results
were obtained when Janus Green B was employed
as a mitochondrial stain. These results indicate a
close phenomenological relationship between
cleavage in E. coli and mitochondrial activity.
(Supported in part by grant-in-aid from the
American Cancer Society.)

29. Response of infertile mice to progesterone
and gonadotrophin. CHARLES A. BaRRa-
CLOUGH (introduced by Rosert D. Tscurreat).
Dept. of Anatomy, Univ. of California, Los
Angeles.

Previous studies have shown that a single sub-
cutaneous injection of 1 mg of testosterone to
5-day-old mice renders these animals infertile
when they reach maturity. Furthermore, the
ovaries of these mice contain few vesicular follicles
and lack corpora lutea. To determine if such
ovaries are capable of responding to gonadotro-
phin, 1 1.u. of chorionic gonadotrophin has been
administered, and the fallopian tubes were ex-
amined for ova 16 hours after treatment. While
all mice ovulated, only 2-3 ova were discernible.
If, however, 1 1.U. of equine gonadotrophin (PMS)
was given 60 hours prior to chorionic gonado-
trophin, 8-9 ova were present at autopsy. Daily
vaginal smears have shown these infertile mice

10 FEDERATION PROCEEDINGS

to be ‘persistent estrus’ animals whereas litter
mate control mice ran a 7-8 day cycle. Daily in-
jections of 0.1 mg of progesterone to these per-
sistent estrus mice induced an average 4-day
cycle in contrast to the average 7-8 day cycle
observed following daily treatment with 0.5 mg
of progesterone. Mating attempts proved unsuc-
cessful. If progesterone (1.0 mg) was injected only
at proestrus, a 6-8 day vaginal cycle was induced.
Four of 6 of these animals, when mated, became
pregnant and delivered 2-3 young/litter within
27 days after being placed with the male. Litter-
mate controls normally delivered 7-10 young 25
days after being placed with the male. These
results indicate that the infertility induced by
androgen is not attributable to unresponsive
gonads but more likely to an imbalance of pi-
tuitary secretion.

30. Histamine-like component of commercial
Pitressin active in releasing ACTH. W. E.
Barrett,* W. W. Swina ez, L. J. BRANNICK,*
S. J. LeBrre* anp A. F. Partow.* Princeton
Univ., Princeton, N. J.

Pitressin is an active ACTH-releasing agent,
but some claim this activity is due to an unknown
contaminant. Histamine has been reported as oc-
curring in the posterior pituitary, hence a search
for its presence in commercial pitressin was
undertaken. Concentrated extracts of Pitressin
prepared by refluxing for 1.5 hr. in 18% HCl at
boiling temperature, the acid removed and the
dry residue taken up in water and neutralized,
contain no pressor activity, instead a histamine-
like component becomes apparent. A 38-fold ex-
tract concentrate given i.v. in 2 ce to atropinized,
etherized cats resulted in a 40 mm Hg fall in blood
pressure. The depressor effect was comparable to
that induced by 4.17 a/kg of histamine base.
Similar extracts of oxytocin exert no effect on
blood pressure. Concentrated pitressin extracts
induced histamine-like responses of guinea pig
ileum. The histamine equivalent of the extracts
was approximately 0.3 a/ce of chamber fluid.
Pyribenzamine abolished the gut response to both
extract and histamine. The histamine-like sub-
stance failed to induce a decline in adrenal as-
corbie acid of 8 hypophysectomized rats, but was
active in releasing ACTH from halved pituitaries
incubated 1 hr. in the Warburg apparatus. Injec-
tion of minute amounts of the incubation fluid
i.v. into each of 16 hypophysectomized rats in-
duced a significant fall in adrenal ascorbic acid.
Incubation fluid from the remaining or control
half of the gland gave negative results in 15
hypophysectomized rats. The ACTH releasing
agent may be histamine combined with a peptid
or amino acid.

Volume 1§

31. Physiological responses during coitus in
the human. Roscor G. BARTLETT AND V. ¢
Bour (introduced by FREEMAN H. Quipy),
Physiology Research Labs., Colton, Calif.
Simultaneous recordings of the heart rate were

made on the human female and male before,
during and after coitus. The results showed g
marked increase in heart rate accompanying the
orgasm in both the male and the female. The
response of the male was more uniform than that
of the female. A striking parallelism between the
responses of the male and the female in a single
test was observed in several of the experiments,
The importance and nature of the mechanisms in-
volved are mentioned. In addition, simultaneous
recordings of the respiratory rate, respiratory
minute volume and respiratory tidal volume were
made on the human female and male before,
during and after coitus. The results showed that
there was a marked increase in the rate and minute
volume in many of the tests. The effect on the
tidal volume was varied depending on the respira-
tory rate. The data strongly suggested that hyper-
ventilation was present during the erotic arousal
and especially during the orgasm. The possible
role of this hyperventilation in the sensory phe-
nomena associated with erotic arousal and orgasm
are discussed.

32. Glycogen particles in rat liver homog-
enates. A. D. Barton (introduced by Dovua.as
E. Smita). Div. of Biological and Med. Research,
Argonne Natl. Lab., Lemont, Ill.

When the microsome fraction from rat liver is
resuspended in sodium desoxycholate (DC) solu-
tion, the large microsome particles are solubilized.
If this procedure is carried out in 0.88 m sucrose,
sedimentation at 100,000 X g then yields a colorless
glassy pellet which contains almost no N or P,
but consists of glycogen (cf. PALADE AND SIEKE-
vitz, Federation Proc. 14: 262, 1955, and Lirrie-
FIELD et al., J. Biol. Chem. 217: 111, 1955); the
glycogen sediments to form a more or less ho-
mogeneous jelly. When this pellet is resuspended,
deposited on grids and shadowed, examination
under the electron microscope reveals blebs of
homogeneous material of low electron density,
with sharp edges and a characteristic flat domed
appearance. The smaller blebs are approximately
circular in outline and apparently represent indi-
vidual glycogen particles originally present in the
homogenate. The larger blebs are irregular in
shape; on resuspension, much of the glycogen is
not dispersed into the individual particles but
remains in the form of small blobs of jelly. In
material not treated with DC, these larger glyco-
gen blebs may contain microsome particles which
became embedded during sedimentation. Micro-
some preparations, with and without DC treat-

valt
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March 1956

ment, have been centrifuged at various gravita-
tional forces and the parts of the pellets which
resuspended most readily have been examined. In
the preparations dried on grids, the individual
glycogen particles associated with the microsome
fraction have been found to range from 300 to
9500 A in diameter. (Work performed under the
auspices of the U.S. Atomic Energy Commission.)

33. Effect of dehydrated plant juices in diet
on Qo, of rat tissue slices. Joun P. Baum-
GgarpT,* G. Cart Rau, THEropore Avyone,*
L. L. Ersenspranpt. Dept. of Pharmacology,
Univ. of Kansas City, Kansas City, Mo.
Sprague-Dawley strain white rats were used in

astudy of the effect of dehydrated plant juice on
Qo.. Friskies Dog Cubes and water comprised the
control diet. Test animals were fed 75% Friskies
and 25% dehydrated plant juice (Verdazyme, sup-
plied by E. H. Pratt, Kansas City, Mo.) plus
water. The dehydrated plant juice preparation
consisted of expressed juice from young cereal
plants desiccated at low temperature under
yacuum. A typical 1 gm. sample showed the
following analysis: vitamin A—529 units, choline—
1.66 mg, inositol—0.05 mg, tryptophane—1.0 ug,
vitamin B,—14.0 ug, Be—33.2 ug, Be—17.0 ug,
By2—0.06 ug, C—1.5 mg, pantothentic acid—48.0
ug, niacin—110 ug, biotin—1.12 yg, folic acid—6.5
ug, plus minerals. The in vivo effect of the modified
diet on oxygen uptake from liver, kidney and
brain slices of 39 young adult rats was determined
using a standard manometric technique. The
number of control vessels varied from 55 to 112
for each tissue and test vessels varied from 40 to
80. Differences in mean Qo, during the first 90-min.
period were not significant. Mean control Qo,
values were: liver 1.77, kidney 3.33 and brain
cortex 1.81. No significant changes in mean Qo,
values were found during the last 90-min. period
except in brain slices. The mean Qo, value for
control brain slices was 1.59 and that of test
animals was 1.72, a 36% higher level.

34. Ventricular electrolyte shift during vari-
ation of contractile force. E. Bay anp I. Bay
(introduced by W. S. W1LpE). Depts. of Pharma-
cology and Physiology, Tulane Univ. School of
Med., New Orleans, La.

The effuogram of O’Brien and Wilde depicted
the K release of systole as a distinct pulse. In
order to investigate whether the release might be
related to electrical or contractile events, we have
tested whether the amount of release is related to
the magnitude of contractile force by altering the
latter by each of 3 strategies: aortic constriction,
l-norepinephrine, Cedilanid D. Contractile force
was measured by a strain gauge arch sutured to
the left ventricle of each of 15 mongrel dogs under

AMERICAN PHYSIOLOGICAL SOCIETY 1]

barbiturate anesthesia, with open chest and
artificial respiration (100% O:2). Rise or fall of
coronary sinus plasma concentration above ar-
terial was interpreted to indicate release or uptake
of ventricular electrolyte in samples for Na, K and
Ca taken every 2 min. Simultaneous samples from
the femoral vein detected electrolyte changes in
other organs. Na and Ca underwent no significant
changes under any of the 3 conditions. Constric-
tion of the thoracic aorta or infusion of /-norepi-
nephrine each increased contractile force of the
ventricle. Plasma K concentration in sinus and
arterial blood rose significantly, but parallel to
each other. No change in ventricle content of K
is implied and the elevation of plasma K evi-
dently came from extracardiac sources. After a
massive dose of digitalis (0.05-0.125 mg/kg Cedi-
lanid D) contractile force increased with increas-
ing doses. Coronary sinus outflow did not change,
or increased slightly. Plasma K concentration in
coronary sinus blood increased significantly above
that in arterial blood, as long as the animal was
not affected by Oz deficiency due to the procedure.
The marked increase in contractile force after
massive digitalis injection might be associated
with increased K release from heart muscle, as
indicated by an inverted A-V difference, but in-
creases of force imposed by other agents are not
accompanied by detectible K shift in the myo-
cardium. (Partially supported by Atomic Energy
Commission contract AT(40-1)-1301, Life In-
surance Med. Research Fund contract G 55-28
and American Heart Assoc., Inc.)

35. Reserpine and reaction to O; at high pres-
sure. JOHN W. BEAN. Dept. of Physiology, Univ.
of Michigan, Ann Arbor.

Our earlier data suggest that the hypothalamus
plays an important role in reactions to O2 at high
pressure (OHP) and more recent evidence from
autonomics study supports this contention. It
seemed of interest, therefore, to determine what
effect reserpine, which is believed to act through
the hypothalamus, might have on the animals’ re-
sponse to OHP. Experiments were carried out on
36 young male rats. Test animals were admin-
istered Serpasil (.07 mg/kg) intramuscularly and
controls on equal volume of physiological saline,
daily for 10 days. On the 10th day the animals
were exposed in pairs to O2 at 90 lb. pressure; CO2
was absorbed and temperature maintained at
28°C. Observations were made continuously
throughout the exposure lasting about 25 min.
After decompression in stages the animals were
killed under pentothal anesthesia and their lungs
grossly examined and weighed. Data on the time
threshold of minimal neuromuscular reactions
were inconclusive but the threshold for severe
convulsions was appreciably shorter and the lung

weight slightly but significantly less in the Serpa-
sil-injected animals. The number of animals
convulsing and the gross appearance of the lungs
were essentially the same in the test and control
groups. Similar doses of Serpasil administered to
control rats in air was without effect on the weight
or gross appearance of the lungs. The body weight
of the tests was about 2% less than that of the
controls on the 10th day. While the data indicate
that Serpasil, as administered in these experi-
ments, provides no significant protection against
O, convulsions they do suggest it may provide a
small degree of protection against pulmonary
edema induced by OHP.

36. Renal clearance studies in Wilson’s dis-
ease. A. G. Bearn, T. F. Yi, A. B. Rirrer-
BAND AND A. B. GuTMAN (introduced by V1n-
cENT P. Doe). Rockefeller Inst. and Dept. of
Medicine, Mount Sinai Hosp., New York City.
The renal defects in Wilson’s disease were

studied by simultaneous renal clearance tech-

niques in 7 patients, 13-53 years of age. All had
symptoms of 3-23 years’ duration, reduced serum
ceruloplasmin and increased urinary copper ex-
cretion, 6 had distinctly reduced serum copper
levels. Cin was normal in the 2 patients with the
shortest duration of overt disease but was re-
dued in the remaining 5 (52.9-84.7 ml/min.).

Cpan was depressed in all cases (214-453 ml/min.).

Tmpan in 3 of 4 cases studied was very low (21.5-

39.2 mg/min.), implying progressive reduction in

tubular excretory capacity. Tubular reabsorptive

mechanisms also were impaired. Excessive amino-
aciduria was present or easily produced in all

cases; in 6 Ca-amino N/Cy, was increased (0.04-

0.17). Cros was uniformly increased (0.18-0.59), in

6 cases fasting Ppos was low (2.5 mg% or less).

Purate Was uniformly low (3.0 mg% in 5 cases).

Curate/Cin consistently was increased (0.17-0.41).

Probenecid,in doses which ordinarily greatly in-

crease Curate/Crn and depress Tmpan, elicited a

negligible increase in Curate/Cra in the 2 cases of

longest duration and slight to moderate increases
in the remaining 5; no striking depression in

Tmpan was observed. Thus, early in the disease

the renal abnormality appears restricted to mild

impairment of renal functions generally ascribed

to the proximal tubules. With progression, tubular
transport systems become increasingly affected
and eventually depression of GFR supervenes.

The evidence supports the view that the renal

disorder in Wilson’s disease is a secondary phe-

nomenon, presumably due to excessive deposition
of copper, and is not the primary metabolic defect.

37. Effect of succinate and fumarate on
ketone production by liver slices from non-
diabetic and diabetic rats. C. H. Brarry,

2 FEDERATION PROCEEDINGS

Volume 16

E. 8S. West* ann R. M. Bocex.* Biochemistry

Dept., Univ. of Oregon Med. School, Portland.

We have demonstrated previously that several
tricarboxylic acid cycle constituents decrease
ketonuria in non-diabetic rats but not in alloxan
diabetic animals (J. Biol. Chem. 190: 603, 1951;
215: 661, 1955). In the experiments reported here
we measured ketone production by liver slices
with and without tricarboxylic acid cycle con-
stituents. The difference in ketone concentrations
in the medium at 15 and 75 min. was taken ag
production. The diabetic rats were used 3-6 wk.
following alloxan administration (fasting blood
sugars above 200 mg %). All animals were fasted
14 hr. In 8 experiments on non-diabetic liver slices
succinate decreased ketone production by 13.64
3.9 (s.e.) ug/100 mg (wet weight), with no change
in the glucose level of the medium. In 9 experi-
ments with diabetic livers, succinate did not
change the production rate of ketones (—2.6+2.2
ug/100 mg) although the glucose concentration of
the medium increased. A marked increase in 0;
consumption with succinate was comparable in
both series. Ketone production following addition
of fumarate changed by —11.3+4.1 ug/100 mg in
8 experiments with normal slices and —12.6+2.1
ug/100 mg in 6 experiments with diabetic slices.
Fumarate caused an increase in glucose concentra-
tion of the medium in both series. Fourteen non-
diabetic and 8 diabetic livers showed no difference
in dry weight, total nitrogen or O2 uptake. How-
ever, liver size relative to non-fasting body
weight increased in the diabetics. (Supported in
part by Natl. Insts. of Health, PHS.)

38. Ventilation-blood flow ratio differences
between the lungs of the normal dog.
A. A. BrcuTeL (introduced by J. F. Mc-
CiENDON). Dept. of Physiology, Hahnemann
Med. College, Philadelphia, Pa.

Direct evidence of regional inhomogeneity of
alveolar air in the anesthetized normal dog has
been collected by simultaneous sampling of end
expiratory gas through catheters placed in the 2
sides of the respiratory tree just below the tracheal
bifurcation. Analytically significant differences
are the rule rather than the exception. Averages
for 2 additional series of experiments confirm the
order of magnitude of differences heretofore
reported (Federation Proc. 11: 10, 1952) both for
animals lying undisturbed on the back throughout
the experiment, and for those whose position was
changed from side to side (as well as back) during
an experiment. With the dog on its back the
differences indicate that the right lung tends to
have a higher ventilation-blood flow (V/Q ratio
than the left (P = .08 for 9 of 11 dogs, .005 for 35
of 48 observations). When the dog is lying on its
right side, the right-lung v/Q ratio superiority

dec
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act
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1951;
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March 1956

persists. When the left side is down, however, the
V/Q ratio favors the left lung. When the left-lying
data are compared with the back-lying, the left
jung V/Q ratio superiority is significant at the
005 level. It is assumed that in the side-lying
position the ventilation of the lower lung is
decreased relative to the upper. The increase in the
left lung V/Q ratio as compared with the right
accordingly indicates a decrease in blood flow in
the left lung when it is down, which is greater (in
proportion) than the decrease in ventilation. When
the right lung is down the changes in ventilation
and blood flow appear to be of the same magnitude
proportionally.

39. Pharmacologic doses of insulin on mouse
liver NPSH. Lyte V. Beck anp ApELE M.
Branconi.* Dept. of Physiology and Pharma-
cology, Univ. of Pittsburgh School of Medicine,
Pittsburgh, Pa.

Binkley and co-workers (J. Biol. Chem. 192: 29)
have reported that high insulin dosage induces
appreciable decrease in the liver glutathione of
fasted rats. In the present experiments male mice
were killed 3-8 hr. after subcutaneous injection of
insulin. For mice killed 4 hr. after injection of
insulin, 0.01 u/gm, or a control solution, the
decrease in liver NPSH found for a) nonfasted
insulin-injected mice was about as great as that
found for b) noninsulin-injected mice fasted for
18 hr. before injection of a control solution;
c) mice, which were fasted and injected with
insulin, exhibited liver NPSH values not much
lower than those exhibited by either a) or b) mice.
Finally, contrary to expectations, d) mice, which
had been fasted and then injected with a mixture
of insulin and glucose to give an insulin dose of
0.01 u/gm and a glucose dose of 1.5 mg/gm.
exhibited very low liver NPSH values, and more
pronounced prostration than c) mice. No dif-
ferences in effects of ordinary and glucagon-free
insulin (Lilly) were noted. The high doses of
insulin required to induce significant decreases in
liver NPSH indicate that this is a nonspecific
stress effect. As previously reported, marked
decrease in mouse or rat liver NPSH may also be
induced by severe trauma, hemorrhage, exposure
to cold (see also Bartlett e¢ al.). (Supported in
part by Natl. Insts. of Health Grant-in-Aid
RG-3740(C2).)

40. Nucleotide polymerase activity in cell-
free extracts of Micrococcus lysodeikticus.
Ro.tanp F. Beers, JR. (introduced by F. O.
Scumirr). Div. of Biochemistry, Masaschusetis
Inst. of Technology, Cambridge.

Aqueous extracts of lysed cells of M. lysodeikti-
cus after fractionation at pH 8.0 with (NH,)2SO,
and acetone demonstrate strong nucleotide poly-

evrrre

AMERICAN PHYSIOLOGICAL SOCIETY 13

merase activity with ADP as the substrate. The
reaction has been followed in tris buffer by pre-
cipitating and washing the product with 10%
trichloroacetic acid and 95% EtOH. The polymer
is determined quantitatively by its absorption
peak at 257 my in tris buffer. The rate of formation
of the product follows zero order kinetics for the
first 15-30 min. The rate is proportional to the
concentration of the enzyme and the substrate,
the latter up to 5 X 107° m. We suggest the follow-
ing unit of enzyme activity: the amount of enzyme
present in 1 ml which will (theoretically) convert
50% of the substrate to the polymer in 1 min. In
the absence of Mg no polymerization occurs. The
rate increases linearly with Mg concentration up
to 1.5 X 10-* m (pH 8.0, 3 X 10-* m ADP), and is
inhibited at higher concentrations. The px opti-
mum = 8.5-9.0, the activation energy = 12 Cal.
The polymer has an absorption spectrum in-
distinguishable from that of ADP. A heat stable
myokinase is present. Mixtures of C"-labeled
AMP and nonlabeled ATP yield a product with a
specific activity 4 that of AMP, indicating that
ADP is the true substrate. The enzyme prepara-
tions contain a residue of nucleic acid amounting
to only a few % of the total polymer formed. Paper
chromatography studies with C-labeled ADP
have yielded results similar to those reported by
Grunberg and Ochoa (J. Am. Chem. Soc. 77: 3165,
1955). (Supported by the Natl. Cancer Inst., Natl.
Insts. of Health.)

41. Lean body weight (LBW) and body weight
from anthropometric measurements.
ALBERT R. BEHNKE. U. S. Naval Radiological
Defense Lab., San Francisco, Calif.

In support of previous studies (Federation Proc.
1954) for estimation of LBW from ‘skeletal’
measurements (SM) are data on 31 Navy men,
M Wig 78.3 (range 52-117), M Hem 178.3 (range
161-201.5). Mean values (nearly identical with M
values on two other independent groups by Hertz-
berg, Daniels, Churchill (Air Force, N = 4050)
and Willoughby (Athletes, N = 52) for individual
SMem were: biacromial, 40.6; chest W, 29.9; bi-iliac,
29.4; bitrochanteric, 32.8; knee-knee W, 20.5; circ
wrist, 17.2 and ankle, 22.5. From the basic formula
LBW = 0.467 (M 2SM)?H, calc M LBW = 63.3x,
(range 47-88). Mean ‘envelope’ measurements
(EM) on the same group of men were: bideltoid,
47.1; and circumferences, chest, 95.1; biceps, 32.0;
forearm, 27.9; buttocks, 97.5; thigh, 57.1; and calf
37.4. The last three values approximate closely
Air Force and Willoughby values but the first four
M values are lower than those on Willoughby’s
athletes. Nevertheless, for the Navy subjects,
Wig calc from Wxg = 0.1375 (M 2 EM)*H was
remarkably close to measured Wxg, i.e. +1.6 (S.E.
of estimate, 2%) with the maximal difference only

14 FEDERATION PROCEEDINGS

3.lkg. Excess weight (W — LBW) in the group
varied from 6 to 29% of W. Density and water
(tritium determinations (by W. Siri, Donner Med.
Lab.) were obtained for comparison with anthro-
pometric data. From different techniques (except
TBH.O) developed by the author over a period of
18 yr. for the estimation of LBW, one may compare
data on the same subject: Wi, 92, LBW 71.8
(sp. gr.); W 83.2, LBW 71.1 (sp. gr.); W 91.6,
LBW 69.3 (antipyrine space 49.9 1); W 93, LBW
69.8 (from basal O2); (W98.2, LBW 71.3 (from
tritium space, 49.6 1, and density by W. Siri);
W 100.7, LBW 68.7 (from tritium space, 48.8 1,
and density by W. Siri); and (anthropometry)
W 100.2, cale W, 99.4, LBW 72.9. Anthropometry
is the simplest procedure for estimation of LBW
and in contrast to previous techniques is appli-
cable both to normal healthy adults and patients
(excluding those with bone lesions).

42. Factors altering capillary resistance after
macromolecular infusions. V.G. BEHRMANN
AND F. W. Hartman.* Dept. of Labs., Henry
Ford Hosp., Detroit, Mich.

In previous reports, a reduced capillary re-
sistance associated with low platelet, prothrombin
and fibrinogen levels and hemorrhagic sequelae
has been demonstrated after infusions of dextran,
PVP, MFG, pectin esters and methy] cellulose,
which is in sharp contrast to the normal findings
following isotonic saline, dog serum or gelatin.
Histologic evidence suggests that liver retention
hampers prothrombin and fibrinogen production.
Dogs injected daily (20 ce/kg) with PVP, dextran,
and methyl cellulose showed a steady decline in
fibrinogen to less than half the initia! level,
whereas gelatin infusions resulted in a fluctuating
fibrinogen level reaching 105-115% of the initial
value on the 21st day. Transient liver retention of
gelatin as opposed to prolonged hepatic storage of
PVP and dextran may explain these results. In
splenectomized dogs platelet levels were higher 24
hr. after gelatin (100-130% of initial level) than
after dextran (60-80%) suggesting a dextran effect
on platelets which allowed removal by the re-
maining RES and/or reduction in platelet forma-
tion by the megakaryocytes. Platelet removal by
the RES must occur after dextran, PVP and
methy! cellulose infusion, for splenectomized dogs
with india ink RES blockade showed prompt
platelet recovery to 100-125% of initial level.
Examination of bone marrow after repeated
infusions of these macromolecular solutions show
marked changes in the megakaryocytes, charac-
terized by swelling and absence of granulation
and platelet formation. Gelatin, however, pro-
duced none of these histologic changes.

Volume 16

43. Olfactory and trigeminal nerve responses
to odors. Lioyp M. BEIDLER AND Doy
Tucker.* Dept. of Physiology, Florida State
Univ., Tallahassee.

Action potentials were recorded from primary
unmyelinated olfactory fibers and from trigeminal
nerve fibers innervating the olfactory area of the
rabbit nasal epithelium. The responses of both
nerves to odors presented to the nose were simul-
taneously displayed on a dual beam oscilloscope.
The olfactory fiber response occurred during each
inspiration and was constant from one inspiration
to the next when the odor concentration was low,
At high odor concentrations the olfactory activity
declined to zero after several seconds of inspira-
tions. When the trachea was cannulated and the
odor was sucked through the nasal passage by
means of a syringe, the olfactory response was de-
pendent upon the flow rate. Abrupt increases in
flow rate that simulated the conditions during a
sniff produced an increase in olfactory response
even though the concentration of the odor entering
the nostril remained constant. The trigeminal
nerve activity increased slowly and was main-
tained during inspiration and expiration. A tri-
geminal nerve response was observed with most
odors that stimulated olfactory receptors.
Thresholds were usually higher than olfactory
thresholds. Activity of both olfactory and tri-
geminal nerves rapidly declined after the stimulus
was removed. Olfactory threshold to amy] acetate
was 0.5-0.8 um mole and trigeminal, 3.0-5.0 uM.
(Supported in part by a grant from the Armour
Research Fndn.)

44. Cardiometer for laboratory and clinical
use. JAMES B. BeE.LL,* E. FuLcHrERo* AND
STEvENS J. Martin. St. Francis Hosp., Hartford,
Conn.

Prompt detection of cardiac arrest is important
in many laboratory studies and imperative in
clinical anesthesia for adequate treatment. While
the Cambridge cardioscope is invaluable, its
expense, sensitivity and bulkiness prevent its
widespread use. Accordingly, a small, inexpensive
cardiometer was developed. The principle of the
apparatus is based on the electronic detection of
the action currents of the heart. It consists of a
small aluminum box, containing a Simpson micro-
ammeter, in a circuit of three stages of resistance
coupled with Class A amplifiers, operated by
subminiature pentodes and by two small batteries
(1.5 and 22.5 v.). Four high value condensors
amplify the low range frequency of cardiac action
currents. An ordinary switch and standard ECG
electrodes complete the apparatus. It was em-
ployed during the past year on man, dogs and
cats for many operative procedures and was
monitored by the Cambridge cardioscope using

the
foll
cal
ani
car
did
cha
dog
sur
car

eXxy
car
not
car

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stre
ene

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tan,
exe!
cha
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The
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whe

the
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ture
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5 to

46.

ume 1§

r»Onses
Dow
| State

rimary
eminal
of the
f both
simul-
scope,
g each
ration
is low.
ctivity
ispira-
nd the
ge by
yas de-
uses in
Tring &
sponse
tering
eminal
main-
A tri-
1 most
»ptors.
actory
id tri-
imulus
cetate
.0 uM.
rmour

inical
* AND
urtford,

ortant
ive in
While
le, its
nt its
ensive
of the
‘ion of
[is of a
micro-
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ed by
tteries
ensors
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| ECG
is em-
zs and
d was
using

March 1956

the standard leads for both instruments. The
following confirmed results were noted: a) Identi-
cal changes in cardiac rate and rhythm of man and
animals were recorded in both the Cambridge
cardioscope and the cardiometer. b) The latter
did not reveal PQRST waves. c) Such identical
changes were also noted in anesthetized man,
dogs, and cats experiencing severe shock due to
surgical trauma, hemorrhage or both, even when
carotid pulse rates and blood pressures could not
be recorded. d) In animals, cardiac arrest induced
experimentally, was promptly detected by the
cardiometer. e) Gross muscular movements did
not effect the recording of cardiac action by the
cardiometer.

45. A human calorimeter. T. H. BENzINGER,
R. G. HuesscHEerR,* Davip MINARD AND CHAR-
LOTTE KitTziNGER.* Naval Med. Research Inst.,
Bethesda, Md.

In a cavity completely lined with a uniform
gradient layer, total rate of energetic output from
asource may be continuously and rapidly recorded
(Rev. Scient. Instruments 20, 849, 1949). Heat given
off into circulating ventilatory or respiratory air
can be measured similarly, with gradient layers
in heat exchanging plates (ibid.). Our human
calorimeter, intended for studies of physiological
temperature control, effects of environmental
stress, human performance and work efficiency,
energetics of metabolism, peripheral hemo-
dynamics and related problems of pharmacology,
pathology and clinical medicine, has 2 compo-
nents: the main shell with 3600, and the venti-
latory heat exchangers with 1344 copper
constantan foil junctions of previous design
(ibid.). The shell, of pure aluminum, is rec-
tangular, 32 X 32 X 84 in., permitting exhaustive
exercise in the supine position. Performance
characteristics are as follows: linear response, 70
uv-sec/cal.+0.8%, independent of location, size,
shape and surface temperature distribution of the
source; half response time of the layer, 4 sec.;
half adjustment of temperature distribution at
800 1/min. flowrate in the main shell, 44 sec. and
in the ventilatory circuit, 64 sec. (nonexpo-
nential); response potential, 70 uv-sec/cal.1%.
The 2 potentials in series represent total energetic
output with +1% error, even under conditions
where a wet source withdraws heat from the shell
which then produces a negative potential while
the circuit is recording more positive heat flow
than the source produces. Environmental tempera-
ture can be set at desired levels from +2 to +82°C.
with an absolute humidity of incoming air from
5to 90 mm Hg.

4. LSD and urinary inorganic phosphate
excretion. JoHN R. BERGEN AND Norman E.

AMERICAN PHYSIOLOGICAL SOCIETY 15

Betsaw.* Worcester Fndn. for Exptl. Biology,

Shrewsbury, Mass.

Urinary phosphate excretion in normal guinea
pigs (Ca. 400 gm) following treatment with lysergic
acid diethylamide (LSD-25) and with ACTH has
been studied in order to compare the responses of
guinea pigs with those of humans treated in a
similar manner. Basal urinary inorganic phosphate
values were determined on 24-hr. urine samples.
After reference values were established, experi-
ments with LSD and ACTH were performed in
random order so as to exciude the possibility of
cumulative drug action. No significant change
from the basal value resulted after treatment
with either 10 y or 25 y LSD. Fifty y depressed
phosphate excretion by 47%, 100 y depressed the
value by 20% and no inhibition of phosphate
excretion was evident when 150 y were ad-
ministered. ACTH administration alone had a
variable effect on phosphate excretion in normal
animals but increased phosphate excretion when
administered to LSD-treated animals. The ad-
ministration of ACTH and LSD simultaneously
blocked the action of LSD, indicating an an-
tagonistic action of the 2 drugs. These results are
in agreement with those reported for human
subjects treated with LSD and ACTH (Hoaatanp,
RINKEL AND Hype. Arch. Neurol. & Psychiat. 73:
100, 1955). (Supported by Contract N6ori-197 of
the Office of Naval Research.)

47. Effect of vagal stimulation and acetyl-
choline on contractility of ventricles and
on coronary blood flow in the dog. Erik
BERGLUND, GUNTHER SCHREINER, FRANK Durr
AND Hans Borst (introduced by Wriuiam H.
Forses). Dept. of Physiology, Harvard School
of Public Health, Boston, Mass.

In dogs anesthetized with morphine and chlora-
lose-urethane, the chest was opened and flow-
meters inserted for measurement of aortic and
left coronary artery blood flow. Pressures were
measured in right and left auricles, pulmonary
artery and aorta. After section of both vagi in the
neck, the right and/or left vagus nerve (distal

_end) were stimulated electrically, producing a

fall in heart rate from 145 to 210/min. to 100/min.
or lower. When the heart rate was maintained at
a constant high level by simultaneous electrical
stimulation of one auricle, vagal stimulation had
no effect on ventricular stroke work or filling
pressures. Peripheral resistance fell slightly or not
at all. Coronary artery flow fell only when arterial
pressure fell. Marked inhibition of atrial contrac-
tion was noted by direct vision. Infusion of acetyl-
choline 22-36 y/kg/min. into the vena cava was
accompanied by marked to moderate systemic
vasodilation and lowering of the ventricular func-
tion curves, i.e. less stroke work was obtained at

16 FEDERATION PROCEEDINGS

a given filling pressure. Heart rate was unchanged
or decreased slightly. Coronary flow per minute
work and per aortic pressure was not increased;
this indicates that acetylcholine did not produce
coronary vasodilation. It is concluded that vagal
stimulation of such magnitude as to lower heart
rate from 145 to 210/min. to below 100/min. did
not liberate enough acetylcholine to produce a
negative inotropic effect on the ventricles.

48. Assimilation and dissimilation of carbo-
hydrates by baker’s yeast with 1-C,,-labeled
glucose. H. L. BerKE* anp A. ROTHSTEIN.
Div. of Pharmacology, Dept. of Radiation Biology,
Univ. of Rochester, Rochester, N. Y.

A study was made of the patterns of assimila-
tion and dissimilation of carbohydrate in yeast
using 1-C,,-glucose. Methods were developed for
the separation and purification of the primary
cellular carbohydrates, trehalose, glycogen,
mannan and glucan, so that tracer techniques
could be applied. Trehalose as well as glycogen
serves as a reserve carbohydrate, increasing dur-
ing fermentation of glucose and decreasing during
fasting. Mannan and glucan, on the other hand,
remain relatively constant in amount. The gly-
cogen incorporates radioactivity at the same rate
as it increases in amount. During a subsequent
period of fasting the rate of loss of radioactivity
is also the same as its rate of disappearance chem-
ically. Thus the glycogen behaves as though the
glucose units last incorporated are the first mo-
bilized with no turnover of the glycogen originally
present. On the other hand, trehalose incorporates
radioactivity both by formation of new material
and by turnover of the total trehalose pool.
Mannan and glucan show a very slow turnover of
Cis. Dinitrophenol in low concentrations reduces
the rate of formation of glycogen and trehalose
from glucose. In higher concentrations it markedly
enhances the rate of dissimilation of trehalose
and has a Tesser effect on glycogen. It has no effect
on the glucan and mannan levels. In the absence
of substrate, the marked breakdown of trehalose
in the presence of DNP, results in the appearance
of equivalent amounts of free glucose in the me-
dium. (Based on work performed under contract
with the U.S. Atomic Energy Commission.)

49. Cortical eye motor fields of the cat. ELLIs
C. Berkowitz aNnD J. TREvoR SILVERSTONE
(introduced by Rosert B. Livineston). Depis.
of Anatomy and Physiology, Univ. of California,
Los Angeles.

In cats with the spinal cord sectioned acutely
at C-1 (encéphale isolé) the cortical eye motor
fields have been mapped. The occipital field in-
cludes most of the occipital lobe (including visual
sensory area J and II) and extends forward to the

Volume 1§

middle of the lateral, suprasylvian and middle
ectosylvian gyri. This area is several times larger
than that reported by Claes (Arch. internat. de
physiol. 48: 238, 1939). The eye movements evoked
were always conjugate and contralateral to the
stimulated side, and had a latency of 10-20 see,
The movements were uniformly smooth through-
out the excursion. Within the occipital field move-
ments predominantly upward or downward haye
been found. The frontal eye field lies just above
and below the cruciate sulcus on the medial sur-
face of the hemisphere, and includes the depths of
that sulcus. This confirms earlier observations by
Livingston (Federation Proc. 9: 395, 1950) and
others. Stimulation here causes ocular deviation
which is conjugate and contralateral to the hem-
isphere stimulated and which has a latency of
15-25 sec. Prior to lateral movement there gen-
erally is pupillary dilatation and mid-positioning,
and the movement itself is saccadic. The mid-
positioning reaction seems to be an integral part
of the total movement. Simultaneous stimulation
of the frontal and occipital eye fields (either ipsi-
or contralateral) demonstrates the dominance of
the frontal field. Evidence is presented which
shows that such interaction occurs at a subcorti-
cal rather than cortical level. (Supported in part
by the Public Health Service.)

50. Human cerebral function: disturbances
in conditioning in patients with cerebral
lesions. Louts BERLIN* AND HARoLp G. Wo Fr.
Depts. of Medicine (Neurology) and Psychiatry,
New York Hospital-Cornell Med. Center, New
York City.

A complex conditioned reaction dependent upon
cerebral hemisphere function was studied to
clarify whether a particular part or the whole
cerebrum is involved in the conditioning process
and to ascertain to what extent the degree of im-
pairment of function is related to mass of tissue
loss. Fifty subjects had intact cerebral hemi-
spheres and 23 had cerebral hemisphere lesions.
Of the latter, 11 had one circumscribed lesion of
known dimensions and location: 6 in frontal lobes,
3 in temporal lobes, and 2 in occipital lobes. Con-
ditioning procedures aimed at establishing stable
positive conditioned responses to a tone; negative
conditioned responses to a light and calibrated
tones; and finally at carrying out the negative
and positive conditioned reactions in sequence.
Results of the procedures: subjects with cerebral
hemisphere damage had difficulty in predictably
relating a symbol to a significant event. Even
after experiencing a positive or negative inte-
grated reaction, individuals with hemisphere
damage had difficulty in stabilizing such a re-
action; but once having established and stabilized
a positive conditioned response to a tone, those

2. 2B
cet
TOD
Bic
Th

(reas

lume 16

middle
3 larger
nat. de
evoked
to the
20 see.
Lrough-
| move-
d have
, above
ial sur-
pths of
ions by
0) and
viation
e hem-
ney of
re gen-
ioning,
e mid-
al part
ulation
er ipsi-
nee of
which
beorti-
in part

yances
rebral
NV OuFr.
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r, New

it upon
ied to
whole
DTOcess
of im-
tissue
hemi-
esions.
sion of
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3. Con-
stable
»gative
brated
gative
juence.
srebral
ictably
_ Even
> inte-
sphere
& Te
bilized
, those

March 1956

with cerebral damage could then more readily es-
tablish a second positive reaction to a different
tone. Both groups readily achieved differentiation
between positive and negative conditioned stimuli
when qualitatively different stimuli were em-
ployed. But, subjects with damaged hemispheres
had the greatest difficulty in maintaining stable
negative and positive conditioned reactions when
2or more stimuli previously linked with negative
and positive responses were presented in sequence;
and repeated failures disrupted previously stable
conditioned reactions. Regardless of site of hemi-
sphere damage, difficulties in establishing stable
positive and negative conditioned reactions were
the same, but proportional to amount of damage,
complexity of adaptations, and probably to rate
at which damage occurred.

jl. Platelets in peripheral circulation of the
hamster (Mesocricetus auratus) (motion

picture). Herspert J. BermMan,* GerorcE P.

FutToN AND Brenton R. Lutz. Dept. of

Biology, Boston Univ., Boston, Mass.

Platelet morphology and demonstration of
platelet function in living blood vessels have been
recorded, and platelets in vitro photographed for
morphological comparison. Platelets in vivo were
disk-shaped on surface view and bayonet-like for
lenticular on side view. Platelet plugs were dem-
onstrated sealing small holes in a vessel wall.
Injury resulted in platelet thrombus formation.
Venous vessels were more susceptible than arterial
vessels. Platelet thrombi formed in injured ar-
teries regardless of the rate of blood flow. After se-
vere injury, arterial flow was completely blocked.
In the arterioles, the higher blood pressures and
faster flow decreased the ease of thrombus forma-
tion in comparison with that of venous vessels.
Platelet thrombi in arterial vessels did not cause
constriction. A film sequence shows how a platelet
coating was formed and maintained. The platelets
adhered individually to the endothelium and to
ther adhering platelets. They also broke away
one by one. An equilibrium between those sticking
and breaking away was reached and in this way
the coating was maintained. Commercial thrombin
and a complex thromboplastin such as Russell’s
viper venom were applied topically to blood ves-
vls and initiated platelet adhesiveness and
thrombi. (Supported by Grant H-902 (c4), Natl.
Heart Inst., PHS, and the Dept. of the Army,
Office of the Surgeon General.)

2. Blood pressure in old hamsters (Mesocri-
cetus auratus). HERBERT J. BERMAN,* BREN-
Ton R. Lutz anp GrorcE P. Futon. Dept. of
Biology, Boston Univ., Boston, Mass.

The blood pressure of the hamster does not in-
tease with age. The blood pressures of 32 ham-

AMERICAN PHYSIOLOGICAL SOCIETY 17

sters 1 yr. and older were compared with the blood
pressures of 52 hamsters 8-12 wk. old. All hamsters
were caged individually, handled frequently and
given Purina laboratory chow and water ad li-
bitum. The blood pressure was determined in-
directly by the cheek pouch cuff method (Federa-
tion Proc. 14: 12, 1955). The arithmetic mean and
standard deviation for the old hamsters were 77.7
mm Hg+13.6 and for the young hamsters, 90 mm
Hg+11.3. By the Pearson 2 X 2 method, the sig-
nificance of the difference between these 2 means
was 5.07 standard deviations. Fifty-nine blood
pressure determinations were made on the old
hamsters over a period of 6 months. The readings
were separated into 3 age groups as follows: 1 to
1.5 yr. (76.3412.3 mm Hg); 1.5 to 2 yr. (79.44+13.5
mm Hg) and over (76.9+14.9 mm Hg). Determina-
tions in the respective age groupings totaled 19,
24 and 16. The length of the period of anesthesia
produced by the same dose of Nembutal (5 mg/100
gm of b. wt. with 1.5 mg increments when needed)
was longer in the old than in the young hamsters
(39.14+23.8 min. as opposed to 28.7+11.3 min.).
Thus, in old hamsters the blood pressure did not
increase but actually decreased, and the length of
the period of anesthesia produced by the same
dose of Nembutal was lengthened. (Supported by
grant H-902 (c4), Natl. Heart Inst., PHS.)

53. Relation of the adrenal to phosphorus
metabolism of bone marrow. IRWIN BER-
MAN,* ABRAHAM EDELMANN AND ALBERT S.
Gorpon. Depts. of Biology, Grad. School of Aris
and Science, New York Univ., New York City,
and Brookhaven Natl. Lab., Upton, N. Y.
This report describes the effects of adrenal-

ectomy and some forms of replacement treatment

upon the incorporation of P® into 4 phosphorus
fractions of rat bone marrow. A significant in-
crease in P® incorporation into the ribosenucleic
acid phosphorus (RNAP) and deoxyribosenucleic
acid phosphorus (DNAP) fractions was observed
in the bone marrow at 1 day after adrenalectomy.
The specific activity of DNAP, RNAP and phos-
pholipid phosphorus (PLP) in marrow was sig-
nificantly decreased in the 5-day adrenalectomized
group. The incorporation of P® into DNA was
still below normal at 10 days after adrenalectomy
although the activity of PLP and RNAP had re-
turned to control levels. By 14 days after adrenal-
ectomy the specific activity of all fractions had
been restored to control values. No changes were
observed in the incorporation of P® into the total
acid soluble phosphorus fraction. One-half mg
doses of hydroxycorticosterone (F) prevented
the marrow changes noted in the 5-day adrenal-

ectomized animals. Four mg levels of F and 0.5

ml doses of lipo-adrenal cortex extract increased

the specific activity of DNAP, but were ineffective

18 FEDERATION

on the other marrow fractions.
tions of these results to the
adrenalectomy and hormonal treatment induce
in the cytology of the marrow will be discussed.
(Supported by the U. 8. Atomic Energy Commis-
sion and the American Cancer Society.)

The possible rela-
alterations which

54. Hypothermia and coronary blood flow.
Rosert M. Berne. Dept. of Physiology, Western
Reserve Univ. School of Medicine, Cleveland,
Ohio.

Experiments were performed on pentobarbital -
ized open chest dogs and on dog fibrillating heart
preparations. In 1 group of experiments the com-
mon left coronary artery or its circumflex branch
was cannulated and perfused via a carotid artery.
Interposed between the carotid and coronary
arteries were a pump perfusion system to main-
tain a constant pressure, an optically recording
rotameter for flow measurements and a small coil
for altering the temperature of blood entering
the cannulated vessel. The apparatus was modi-
fied for the fibrillating heart in that a donor dog
furnished the arterial blood for the coronary per-
fusion. A reduction of the temperature of the blood
entering the cannulated coronary artery of 10-15°C
produced an increase in coronary flow. This in-
crease in flow was reversible and was not asso-
ciated with consistent changes in myocardial
oxygen consumption. A positive potassium balance
occurred in the heart during periods when the
coronary arterial blood was cooled. In a 2nd group
of experiments phasic blood flow was measured by
means of a bristle flowmeter in the left circumflex
coronary artery of dogs subjected to generalized
hypothermia. The coronary phasic flow curve
showed a continuous rise throughout most of
diastole. The progressive increase in flow in the
face of a declining aortic pressure is related to
the extremely slow rate of relaxation of the ven-
tricles and consequently is a. function of the
ventricles and consequently is a function of the
gradual diminution in extravascular compression.

55. Influence of adrenal medulla on periph-
eral action of exogenous’ thyroxine.
Haroitp BERNSTEIN,* Maurice 8. GoLpstTEIN
AND EstTE.LLE R. Ramey. Dept. of Physiology,
Univ. of Chicago, Chicago, Il.

The rise in basal metabolic rate evoked by the
administration of exogenous thyroxine can be
inhibited by simultaneous treatment with au-
tonomic blocking agents like Dibenzyline. The
blocking drugs do not appear to affect the basal
metabolic rate maintained by the animal’s own
thyroid hormone but prevent the increased oxygen
consumption of ‘hyperthyroidism’ produced by ex-
cessive amounts of injected thyroxine. The role
of the adrenal medullary hormones in this phe-

PROCEEDINGS

Volume

nomenon was studied; 75-gm male rats of the
Sprague Dawley strain were surgically medullee.
tomized by mechanically crushing the adrenal
gland. At least 5 wk. were allowed for regeneration
of the adrenal cortical tissue. At the time of the
experiments the animals showed normal streg
responses, growth and activity on the regular
laboratory diet and tap water ad libidum. Autopsy
revealed well regenerated cortical tissue and
complete absence of medullary tissue. The medul-
lectomized rats and normal controls were fasted
for 30 hr. and their oxygen consumption deter.
mined. This was found to be in the order of 6.9-7,7
1. of oxygen/m?/hr. There was no significant dif-
ference between the experimental animals and
their controls. The rats were then fed ad libidum
for 24 hr. and then injected subcutaneously with
1.5 mg/kg of Na-1-thyroxine. A further period of
24 hr. of ad libidum feeding followed this treat-
ment and the animals were then fasted for 30 hr,
and the oxygen consumption determined. The con-
trol animals responded to the thyroxine therapy
with a rise in oxygen consumption up to 99
1/m?/hr. The medullectomized animals showed no
significant increase of oxygen consumption over
their initial values despite the administration of
exogenous thyroxine. It appears that the medul-
lary hormones play some role in the response to
injected thyroxine but are not essential for the
maintenance of normal basal metabolic rates.

56. Acetylcholine esterase as_ trigger for
transmembrane reaction. R. H. BEutNen,
Des Moines Still College, Des Moines, Iowa.
Nachmannsohn has demonstrated the indis-

pensability of acetylcholine esterase for nerve

function, electric organ, etc., but the underlying
mechanism remained unexplained. Acetylcholine
was assumed to cause the action potential, but
how can the destruction of this allegedly electro-
genic substance have such an effect? The answer
may be that acetylcholine splitting is coupled with
phospholipid splitting. This splitting can be shown
to produce an emf of <0.1 v., nearly equal to the
action potential, also in the right direction in-
asmuch as the negative pole is on the side of the
decomposed lipid, relative to the undecomposed
lipid (in a suitable setup). Transient, reversible
phospholipid splitting has been shown to be es-

sential for nerve activation (Federation Proc. 13:

13, 1954). Splitting starts at the outer membrane

contact surface (producing the upstroke of the

spike), then extends across the membrane to pro-
duce the downstroke, because the potential dif-
ference at the inner membrane contact surface
is in the opposite direction. A single reaction (the
transmembrane reaction) can thus produce both
up- and downstroke. This transmembrane re-
action (phospholipid splitting) may be initiated

curr
tery
tent
cont
This
the

whe:
men
rest
lipid
4 po
is fc
now
the x
The
can

cal ri
the |

lume If

| Of the
edullee.
adrenal
1eration
e of the
1 stregg
regular
\utopsy
ue and
-medul-
> fasted
1 deter-
f 6.9-7.7
ant dif-
als and
libidum
sly with
eriod of
s treat-
r 30 hr.
‘he con-
therapy
to 99
»wed no
on over
ition of
medul-
onse to
for the
tes.

er for
UTNER,
Towa.
. indis-
r nerve
lerlying
choline
ial, but
electro-
answer
ed with
2 shown
1 to the
‘ion in-
» of the
mposed
versible
» be es-
roc. 13:
mbrane
of the
to pro-
ial dif-
surface
on (the
ce both
ane re-
ritiated

March 1956

by an enzyme-activating ion driven into the mem-
brane by the stimulating current (the Gerard
principle). That the esterase triggers phospho-
pid splitting is made likely by the fact that this
splitting is accelerated by the addition of more
acetylcholine. However, this can occur only if the
esterase is present in excess as, e.g., in auricular
muscle. (Supported by a research grant from the
Natl. Heart Inst.).

3]. Mechanism of pacemaker and receptor.
R. H. Beutner. Des Moines Still College, Des
Moines, Iowa.

Lillie’s theory explains impulse propagation
through local electric circuits between positive
and negative regions of the neuron. A positive
potential difference is maintained on the outer
membrane contact surface by means of oxidative
processes (BEUTNER AND LozNeER. Protoplasma
19: 370, 1933). This positive potential difference is
transiently abolished by phospholipid splitting
(the transmembrane reaction; see preceding ab-
stract). However, if one region of the membrane
is devoid of oxidative enzymes, no surface oxida-
tion will occur. Consequently, no positive po-
tential difference is formed on the membrane in
this region, but the potential difference remains
negative. Such a permanently negative potential
difference adjacent to a conducting (positive)
membrane will have the following effects: 1) it
will block conduction by abolishing local circuits,
but nevertheless 2) will produce recurrent electric
waves which travel along the nerve. These re-
current waves originate from the diminutive bat-
terry formed by the positive and negative po-
tential differences lined up along the membrane
contact surface. A local circuit is thus established.
This circuit elicits a transmembrane reaction in
the adjacent region and a traveling impulse
whereby the positive potential difference on the
membrane surface is transiently abolished, to be
restored during the refractory period by phospho-
lipid resynthesis. The membrane now has again
a positive potential difference whereby a battery
is formed sending forth another impulse. From
now on this process repeats itself. This might be
the mechanism of a pacemaker as in the sinus node.
The permanently negative potential difference
tan be shown to be readily influenced by potent
drugs, hence may be regarded as a pharmacologi-
tal receptor. (Supported by a research grant from
the Natl. Heart Inst., PHS.)

%. Mechanism of the Branham-Nicoladoni
sign, bradycardia following closure of an
arteriovenous fistula. Donat M. Bruia*
AND Oscar W. SHap.ie. Dept. of Physiology,
Univ. of Louisville School of Medicine, Louis-
ville, Ky.

AMERICAN PHYSIOLOGICAL SOCIETY 19

Because of failure to elicit the Branham-
Nicoladoni sign in dogs anesthetized with barbi-
tal, all dogs were premedicated with morphine
sulfate and anesthetized with chloralose. Arterial
blood pressures and/or heart rates were recorded
electrocardiographically or with a strain gauge
manometer. After in vivo heparinization, an ar-
teriovenous fistula was constructed connecting
both femoral arteries to a femoral vein. In 3 dogs
a bradycardia, initially obtained upon closure of
the fistula, was abolished by successful carotid
sinus denervation. However, in 2 dogs the brady-
cardia was not abolished by successful bilateral
carotid sinus denervation. It was speculated that
this might be due to intact aortic arch stretch
receptors and experiments were done to exclude
these reflexes while leaving the right cardiac vagus
intact. In the 3 dogs of this group, successful de-
nervation of the aortic depressor and carotid sinus
receptors completely abolished the bradycardia.
An occasional dog under chloralose anesthesia
failed to show a bradycardia following closure
of the arteriovenous fistula. However, these dogs
also failed to show reflex bradycardia following
manual stimulation of the carotid sinus or aortic
arch stretch receptors. These experiments indi-
cate that the Branham-Nicoladoni sign is a re-
flex bradycardia, whose afferent limb lies in the
stretch receptors on the arterial side of the circula-
tion. (Supported by Louisville and Ashland Heart
Assocs.)

59. Depressed ventricular function in the
dog during acute cardiac tamponade
treated by the administration of aramine
and wyamine. J. T. Brinton, W. L. Morean,
S. J. SaRNorF AND G. H. Wetcu (introduced by
M. O. Lee). Lab. of Cardiovascular Hemody-
namics, Natl. Insts. of Health, Bethesda, Md.
Simultaneous right and left ventricular func-

tion curves were done in 6 dogs with complete

circulations using the method of Sarnoff and Berg-
lund (Circulation 9: 706) to study the effect of
sympathomimetic drugs on the heart and circula-
tion during acute cardiac tamponade. In 5 dogs,
after 1 or more control curves were obtained,
tamponade of the heart was produced by injecting
an average of 5.6 cc/kg of saline into the peri-
cardial sac. In one dog the pericardium was con-
stricted by sutures. In every instance, the ap-
parent ventricular function curve was markedly
depressed during tamponade. Aramine was then
given in doses of 0.05 mg/kgm i.v. in 5 dogs, and
Wyamine, 15 mg was given to 1 dogi.v. Following
this the apparent right and left ventricular func-
tion curves were significantly elevated in all
animals. This increase in ventricular stroke work
at any given atrial pressure was independent of
changes in pulse rate. After the tamponade was

20 FEDERATION
released, a post control curve was obtained which
was higher than the pretamponade curve in all
animals except one. It is concluded that Aramine
and Wyamine improve ventricular function,
enabling the heart to do more work without an
increase in diastolic fiber length when the latter
is limited by tamponade. These data suggest
that such drugs may be of value as a holding
maneuver in acute clinical tamponade.

60. Efferent discharge in brain stem during
shivering. Lucy Brrzis (introduced by V. E.
Hat). Dept. of Physiology, Univ. of California
at Los Angeles, Los Angeles.

Preliminary microelectrode studies have been
made of the efferent discharge recorded from
within the previously determined central shiver-
ing pathway. Oscillographic records on film were
obtained using metal microelectrodes of 10-25 u tip
diameter inserted into the brain stem of nembu-
talized cats. The recording site usually lay in the
upper midbrain dorsolateral to the red nucleus.
A discharge of spikes was seen whenever the cat
shivered, and disappeared when shivering ceased.
The frequency of firing of the individual units fell
in the range of 12-23/sec. They were shown to be
unrelated to the respiratory or cardiac rhythms
and were not movement artifacts, since they re-
mained after muscular contractions were abolished
with curare. In other experiments, the electrode
was isolated from afferent influence by making
appropriate mid-sagittal and transverse cuts.
Units recorded from such a preparation, under
curare, were considered to be shivering efferents.
The frequency of the units was gradually de-
creased when the cat was immersed in a hot-water
bath, usually disappearing completely within a
few minutes. The return to an ice bath caused
activity of the units to reappear. The body tem-
perature of the cat did not change appreciably
during this sequence of events but a control re-
cording was made a few millimeters dorsal to the
pathway to check the possibility of a direct tem-
perature effect on the neurons. The discharge in
this control site was entirely unaffected by warm-
ing or cooling the cat.

61. Transient occlusion of renal artery on
patterns of solute excretion. WiLiiam D.
BuakeE. Univ. of Oregon Med. School, Portland.
These experiments were carried out as part of

an attempt to determine whether or not all

nephrons of the kidney act in identical fashion.

Studies were done on pentobarbital-anesthetized

dogs receiving hypertonic sodium chloride and/or

glucose solutions intravenously at 14 ml/min.

Animals were prepared by ligating the left ureter,

cannulating the right and looping a thread around

PROCEEDINGS

Volume ig

the right renal artery. Urine collection periods
were of 15 or 30 sec. duration and clearances of
creatinine, PAH, sodium, potassium and chloride
obtained. Ten to 20 sec. occlusion of the rena]
artery (by tension on the thread) led to a sharp
reduction in urine flow and all clearances. The
total deficits (i.e. before clearances returned to
control values) in clearances of creatinine, PAH
and potassium were approximately equal. Hoyw-
ever, urine flow and sodium and chloride clearance
deficits were considerably greater than that for
creatinine, indicating continued reabsorption of
water and sodium chloride during the period of
cessation of renal blood flow and glomerular
filtration. On occasion, continued secretion of
potassium could be demonstrated by a clearance
deficit of potassium less than that of creatinine,
These phenomena were confirmed by the frequent
fall in concentration of sodium in urine and rise
in urinary potassium concentration that preceded
in time the increase in urinary creatinine con-
centration. The patterns of concentration change
either confirm the distribution and separation of
discrete renal processes along the length of all
tubules or indicate that these processes occur in
structurally and functionally different tubule
populations.

62. Os dependence of hemolysis by ultraviolet
light. H. F. Brum anp Joun S. Cook. Nail.
Cancer Inst., Natl. Insis. of Health, and Dept.
of Biology, Princeton Univ., Princeton, N. J.
Hemolysis of rabbit erythrocytes is brought

about by radiation from 3 spectral regions of the

ultraviolet; apparently involving 3 different
mechanisms, two of which are O2 dependent: 1) by
wavelengths longer than 0.32 u, e.g. from a fluores-

cent lamp, ‘Black Lite’ type, with peak at 0.36 p.

Very high dosage is required for this effect, which

is Os-dependent; and seems best explained as

photosensitized oxidation (photodynamic action).

Protoporphyrin may be suspected as the light

absorber. 2) By wavelengths between 0.32 uw and

0.20 u, from an intermediate pressure mercury are

(atmospheric pressure) under ordinary operating

conditions; this effect is independent of Os». Pro-

tein of the cell membrane seems the most likely
light absorber. 3) By wavelengths shorter than

0.2 u, from a low pressure mercury are with quarts

envelope; presumably these come principally from

the resonance lines at 0.186 u. This hemolysis is

Oo-dependent. O2 itself seems the most likely light

absorber, since replacing air with a column of N:

greatly reduces the hemolytic effect.

63. Interaction of myosin with ATP. J. J.
Buium anp M. F. Morates. Naval Med. Re-
search Inst., Bethesda, Md.

Some years ago we applied the Zimm extrapola-

wat
ins
am
anc
and
gas
juic
juic
juic
exer
juic
ting
or ¢
can!
Gas

crea

lume 1§

periods
nces of
hloride
e renal
a sharp
28. The
‘ned to
2, PAH
. How-
2arance
hat for
tion of
riod of
nerular
tion of
-arance
itinine,
equent
nd rise
eceded
1€ con-
change
tion of
. of all
ecur in
tubule

aviolet
. Natl.
1 Dept.
NE
rought
; of the
ifferent
b: 1) by
fluores-
0.36 p.
, which
ned as
ction).
e light
> w and
ury are
erating
)o. Pro-
5 likely
r than
quartz
ly from
lysis is
ly light
n of N:

Ie
ed. Re-

rapola-

March 1956

tion to data on ATP-induced changes in the light
scattered by solutions of 5-hr. myosin in 0.6 m
KCl buffered at pH 7.0. We concluded that ATP
deformed, but did not dissociate, the myosin
particles. Recently, Gergely reinvestigated this
problem, using the same theoretical analysis but
introducing several technical improvements (sili-
cone-coated glassware, narrow slits, etc.); in
contrast with our conclusion, Gergely reports that
ATP dissociates the myosin. Adopting his tech-
niques, we can confirm both our earlier experi-
ments and Gergely’s. Thus the conflict is probably
not due to unreliable experimentation or dif-
ferences in instrumentation (indeed, we under-
stand that Gergely himself has also confirmed us
in unreported experiments). At this juncture the
essential difference in procedure seems to be that
in our experiments solutions of different concen-
tration are obtained by dilution of a stock solu-
tion clarified by high speed centrifugation, while
in Gergely’s experiments the solutions are indi-
vidually clarified. Gergely’s procedure avoids ag-
gregates which conceivably might form prior to
dilution and perhaps from the dilution process.
Nitrogen analyses indicate that high speed centrif-
ugation reduces the protein concentration by
210%. An examination of multicomponent light
scattering theory suggests that this loss would not
account for the difference between the two experi-
mental results. Pending the completion of further
experiments, we suspect that when myosin is ag-
gregated by firm linkages the inflationary stress
caused by ATP adsorption results in lengthening,
whereas disaggregation ensues if the myosin is
held together by weaker links.

64. Effect of human gastric juice on blood
coagulation and clot lysis. Trsor Bop1, C. W.
Wirts AND R. T. Carrout (introduced by M.
H. F. FrrepMan). Div. of Gastroenterology and
Charlotte Drake Cardeza Fndn., Jefferson Med.
College and Hosp., Philadelphia, Pa.
Coagulation of blood and human gastric juice

was studied using fasting, posthistamine, post-

insulin and buffered specimens. A _ standard
amount of blood and digestive juice was incubated
and the time of complete gellation measured. Free
and total acidity, pH, pepsin, viscosity of the
gastric juice and pu of the mixture of blood and
juice were determined. Posthistamine gastric
juice always prevented clotting and postinsulin
juice almost always. The pu of the mixture did not
exceed 5.8 in both series. When fasting gastric
juice was mixed with blood in equal portions, clot-

ting was observed if the px of the mixture was 6

or above. Fasting specimens of juice had signifi-

cantly higher viscosities than after histamine.

Gastric juice buffered with milk, cream, milk and

cream, milk protein-dextrimaltose-dextrose, soy

AMERICAN PHYSIOLOGICAL SOCIETY 21

flour, soy oil and dextrose mixture, and aluminum
hydroxide magnesium, hydroxide gel facilitated
clotting in all instances. Clots derived from blood
and gastric juice mixture retracted slightly but
offered greater resistance to deformation than
controls. The sectioned stained clots contained
elements from gastric juice (mucoproteins?).
Disruption of clots by gastric juice appeared to
vary in relation, mainly to the px of the gastric
juice and erythrocyte content of the clot. Buffer-
ing with milk and milk products failed to prevent
lysis, but giving large amounts of antacid gel or
small doses combined with proteolysates did pre-
vent it.

65. Effect of heavy particles from the B
(n, a) Li’ reaction on acute mortality in
the mouse. V. P. Bonp anp O. D. Easterpay
(introduced by E. P. CronxiTe). Med. Dept.,
Brookhaven Natl. Lab., Upton, L. I., N. Y.
The 28-day mortality rate was determined for

female CFI mice given B"-enriched borax intra-
venously, and exposed 5 min. later to graded doses
of total-body thermal neutron radiation in the
medical facility of the Brookhaven research re-
actor. The LDs for animals given 254 B'/gram
of mouse was 1.42+0.11 X 10'? n/cm? sec., com-
pared with 4.78+0.14 X 10% n/cm? sec. for
exposure to reactor radiations alone. The LDg
for cobalt 60 gamma radiation was 877+31r. The
rem dose rate due to the heavy particles from
boron capture was determined by subtracting that
due to the reactor radiations alone from the total
observed rem dose rate. The average rep dose rate
from boron capture was calculated, assuming the
mouse to be a ‘thin foil’, from the average tissue
boron content, the slow neutron capture cross
section for boron, the measured thermal neutron
flux and the energy release per neutron capture.
The total energy liberated by both the alpha par-
ticle and the recoil Li’ nucleus, a total of approx-
imately 2.4 mev, was used in the rep calculation.
The relative biological effectiveness (apparent
RBE) for acute mortality was found to be 1.7
compared to Co gamma radiation, and 1.1 com-
pared with 250 kvp x-radiation. A marked tend-
ency to 3- and 4-day deaths was observed in the
animals receiving boron capture irradiation, as
opposed to predominantly 9 -to 12-day deaths
following x-, gamma or thermal neutron radiation
alone. The survival times were compared, with
the several radiations noted, at dose levels that
resulted in approximately the same 30-day mor-
tality rate.

66. Abdominal receptor site for emetic action
of x-radiation. H. L. Bortson. Dept. of Phar-
macology, Univ. of Utah College of Medicine, Salt
Lake City.

22 FEDERATION PROCEEDINGS

Cats were subjected to whole-body, high-energy
X-irradiation with a dose of 5500 roentgens (r)
given at the rate of 180 r/min. This dose was pre-
viously established in this laboratory consistently
to cause vomiting within 10 hr. from the start of
irradiation. The following facts lead to the con-
clusion that the receptor site which initiates the
emetic response is located in the abdomen. /)
Shielding the abdomen prevented radiation-in-
duced vomiting whereas shielding the thorax and
head did not. 2) Chronic supradiaphragmatic
vagotomy combined with cord transection at T3,
in 5 cats, invariably prevented the vomiting re-
sponse to radiation, although veratrum still
evoked emesis. 3) Chronic supradiaphragmatic
vagotomy combined with dorsal rhizotomy in-
volving segments T6-T10, in 6 cats, invariably
prevented the vomiting response to radiation,
although lanatoside C still evoked emesis. Vagot-
omy, either alone or in combination with a)
medullary emetic chemoreceptor trigger zone
ablation, b) celiac ganglionectomy or c) lower
thoracic and abdominal sympathectomy, did not
predictably prevent radiation-induced vomiting.
Dorsal rhizotomy alone did not prevent emesis.
It is evident from these findings that the vomiting
resulting from x-irradiation is initiated in the
abdomen, that the afferent impulses traverse
both the vagus and the dorsal roots, that either
of these pathways may suffice for elicitation of the
response, and that the segmental pathway in-
volves innervation independent of the sympa-
thetic distribution. (Supported by grant No.
B-491, Natl. Insts. of Health, PHS.)

67. Responses of human adductor pollicis to
indirect paired stimuli. Stetta BorTeHo,
Rueta Apams* aNd LEON CANDER.* Dept. of
Physiology and Pharmacology, Grad. School of
Medicine, Univ. of Pennsylvania, Philadelphia.
When the ulnar nerve was stimulated at the

elbow with supramaximal percutaneous paired

square wave electrical stimuli (conditioning-test
shock intervals varying from 1 min. to 0.25 msec.),
changes in action potential areas and maximal
developed isometric tension (simultaneously re-
corded from the adductor pollicis) indicated that
in each subject: /) a range of refractory periods
existed, the upper and lower limits of which we
call the long and short refractory periods respec-
tively. This observation may indicate that groups
of nerve-muscle units have different refractory
periods. 2) The value for short refractory period
was the same whether it was determined from
changes in electrical or mechanical activity; it
corresponded to the longest conditioning-test
shock interval when paired stimuli produced the
same response as did a single stimulus. 3) The
value for long refractory period when determined

Volume 15

from changes in electrical activity differed from
that obtained by measuring tension changes. Since
decreases in action potential areas may result from
algebraic summation when the two responses are
partially superimposed while the number of active
units, as indicated by tension, is unchanged, the
long refractory period cannot be determined from
changes in electrical activity. As determined by
measuring tension changes, mean long and short
refractory periods (in msec.) were (8 women aged
21-23 years) 2.48+SE 0.33 and 1.08+SE 0.08 and
(5 women aged 71-86 years) 5.104+SE 0.51 and
0.844+SE 0.04 respectively. Differences between
age groups are statistically significant (P =
<0.05).

68. Apparent Michaelis-Menten constants
obtained from steady state length measure-
ments on the glycerol-treated muscle fiber
ATP system. WILLIAM J. BOWEN AND JACOB
J. Buum. Natl. Insts. of Health and Naval Med.
Research Inst., Bethesda, Md.

When adenosine triphosphate (ATP) is added
to glycerol-extracted psoas fibers, the ATP is
dephosphorylated and the fibers shorten. It was
previously reported that the extent of shortening
varies directly with the concentration of ATP
and not with the amount of ATP split (Bowen,
Am. J. Physiol. 179: 620, 1954). The extents of
shortening have been treated quantitatively by
the method used to estimate Michaelis-Menten
constants for the interaction between myosin and
ATP as studied by light scattering (Buum. Arch.
Biochem. 55: 486, 1955). Michaelis-Menten con-
stants obtained from length measurements (K;)
were compared with constants obtained from de-
phosphorylation rate measurements (K.). Ky, in-
creases linearly as [KCI]-! decreases, and is higher
with added Mgt* than without. There is a linear
correlation between the rate of shortening of
myosin threads and Ky, of psoas fibers at different
[KCl]. At 0.3 M KCl, px 7.0, for fiber bundles 300-
700 » wide, Ky was about 10 times K,. Ky increased
as fiber-bundle width decreased from 600 to 150g.
K, remained constant in this range, but increased
about 10-fold when fibers of 2 4 were studied. Since
Ky, is about 10 K, in the 150-600 » range and K,
at 2 is about equal to Ky at 150 u, it is probable
that Ky, is greater than K, for any fiber dimension,
and that this difference is not a result of diffusion.

69. Life span of the nucleated erythrocyte
with C™“, Kirkianp C. Brace Aanp Paut D,
ALTLAND (introduced by H. Sprcut). Natl.
Cancer Inst. and Natl. Inst. of Arthritis and
Metabolic Diseases, Bethesda, Md.

We have reported (Am. J. Physiol. 183: 91,

1955) that the life span of the box turtle erythro-

cyte exceeds 350 days. Since the reutilization of

th
lak
val

the
Sy
lab
ery

she
Th
roc
mu
zat
lon
an
ery
stu:
bee
mal
tivi
afte
rem

ct
F
with
were
brar
with
Tem
The
by r
appl
and
simu
curr
from
hega
tenti
rent.
cond
and
duet:
and
labor
cours
Assu:
ance
tivel)
hear]
indep
time |
This
high

ume 1§

| from
Since
t from
es are
active
d, the
1 from
ed by
short
1 aged
8 and
1 and
tween
(P =

tants
sure:
» fiber
JACOB
| Med.

added
rP is
[t was
tening
_ ATP
OWEN.
nts of
ly by
[enten
in and
Arch.
1 con-
3 (Kx)
ym de-
Ky, in-
higher
linear
ing of
ferent
os 300-
reased
150 pw.
reased
. Since
nd K,
obable
nsion,
fusion.

rocyte
ut D,

Natl.
is and

33: 91,
y thro-
tion of

March 1956

the methyl,carbon of the glycine-2-C™“ used to
label the cells remains a critical factor in the
validity of this determination, we have used the
same method to measure the known life span of
the bird erythrocyte. Shemin (Cold Spring Harbor
Symposia Quant. Biol. 13: 185, 1948) using glycine
labeled with N* found the life span of the hen
erythrocyte to be 28 days. Our analysis of the
specific activity of the Cin the duck erythrocytes
shows that the life span is approximately 42 days.
This data, plus additional results on duck eryth-
rocyte longevity using Cr! (personal com-
munication F. G. Ebaugh) suggests that reutili-
zation is probably not an important factor in
longevity studies of nucleated erythrocytes. In
an effort to establish the life span of the nucleated
erythrocytes of reptiles and amphibians, the turtle
studies are being continued and experiments have
been set up to study the longevity of toad (Bufo
marinus) erythrocytes. At this writing the ac-
tivity of the turtle cells remains well sustained
after 500 days and the activity of the toad cells
remains undiminished after 270 days.

70. Repolarization of frog ventricular fibers
in low sodium. ALLAN J. Brapy* anv J.
Water Woopsury. Dept. of Physiology and
Biophysics, Univ. of Washington School of Medi-
cine, Seattle.

Frog ventricles were cannulated and perfused
with solutions in which various amounts of NaCl
were replaced by choline chloride or sucrose. Mem-
brane resting and action potentials were recorded
with floating intracellular ultramicroelectrodes.
Temperature was maintained between 17-19°C.
The entire ventricle was excited simultaneously
by rectangular current pulses of 1 msec. duration
applied through electrodes in the input cannula
and the external bath at a rate of 15-30/min. In
simultaneous excitation of all fibers, longitudinal
current flow is zero. This eliminates distortion
from adjacent fiber activity and also makes the
negative of the time derivative of the action po-
tential proportional to net membrane ionic cur-
rent. By assuming that the sum of the Na and K
conductances is constant during repolarization
and diastole and equal to total membrane con-
ductance (WEIDMANN, J. Physiol. 115: 227, 1951,
and preliminary experiments conducted in this
laboratory), it is possible to compute the time
course of the Na and K currents and conductances.
Assuming membrane capacity and total conduct-
ance to be 10 wf/em*? and 500 umhos/cm?, respec-
tively, the calculated Na and K conductances are
nearly proportional to membrane potential and
independent of external Na concentration and
time except for the early period of repolarization.
This relationship fails in combined low Na and
high K solutions. Thus it appears that there is

Syren

AMERICAN PHYSIOLOGICAL SOCIETY 23

some interaction between membrane Na and K
currents. Other findings are that the action po-
tential duration equals a constant times the ex-
ternal Na. Fibers became inexcitable in 15%
normal Na. Overshoot varies directly with the
Na potential. (Aided by Grant B-462 from the
Public Health Service and the State of Washington
Research Fund for Biology and Medicine.)

71. Hemodynamic factors modifying the re-
lationship between ventricular filling pres-
sure and myocardial oxygen consumption.
E. Braunwa.p,* S. J. Sarnorr, R. B. Cass,*
W. N. Srarnsspy* anp G. H. Wetcu.* Lab. of
Cardiovascular Hemodynamics, Natl. Heart Inst.,
Bethesda, Md.

A preparation was devised in which aortic pres-
sure, cardiac output and heart rate could be in-
dependently maintained constant, or controllably
varied over wide ranges in a dog with a complete
circulation. A cannula in the descending aorta led
the blood through a Starling resistance into a
reservoir. A side-arm proximal to this resistance
led blood through a rotameter to the left main
coronary artery. Blood was pumped from the
reservoir into the descending aorta and brachio-
cephalic artery. Pressures were recorded in RA,
PA, LA and aortic arch simultaneously with con-
tinuous recording of total systemic blood flow
(electroturbinometer), left main coronary artery
flow (rotameter) and heart rate (Waters cardio-
tachometer). Periodic simultaneous sampling of
arterial and coronary sinus blood was performed.
This preparation was employed for re-examining
the relationship between myocardial O:2 con-
sumption and diastolic fiber length (as indicated
by ventricular filling pressure). At a constant
heart rate it was possible to maintain filling pres-
sure constant by varying aortic pressure and
stroke volume reciprocally over wide ranges. At
these constant filling pressures, striking changes in
myocardial O2 consumption were consistently ob-
served; O2 consumption was greatest at high aortic
pressures and low stroke volumes, and fell pro-
gressively as aortic pressures fell and stroke
volumes rose. The data obtained, therefore, are
not consonant with the view, obtained from the
heart-lung preparation, specifically relating myo-
cardial O2 consumption to end-diastolic filling
pressure.

72. Comparative effects of l-epinephrine and
l-norepinephrine on portal pressure of
dogs. SHerta M. Briscoe (introduced by
Durwoop J. Smitu). Dept. of Pharmacology,
Univ. of Vermont, Burlington.

Under certain conditions epinephrine causes

a rise of portal pressure in the dog (CHAKRAVARTI

AND Tripop, J. Physiol. 97: 316, 1950). The effects

of epinephrine and norepinephrine on the portal
pressure have now been compared. Dogs were
anesthetized with pentobarbital. A polyethylene
catheter was inserted into the hepatic portal vein
through the splenic vein, and portal pressure was
recorded with a water manometer. Central venous
pressure was also recorded in some experiments.
Injections were made into the descending aorta at
the level of the coeliac artery by means of a thin
catheter inserted through the left carotid. Epi-
nephrine bitartrate was injected in doses from
0.26 wg/kg-18.3 ywg/kg. Equiponderant or equi-
molecular quantities of norepinephrine were in-
jected. In 15 out of 17 experiments the rise in
portal pressure following epinephrine was greater
than the rise following norepineprine. The onset
of the rise in response to epinephrine was earlier
than that following norepinephrine, and the pres-
sure rose more steeply. In four experiments the
rise was preceded by a fall. Norepinephrine pro-
duced a much greater pressure decrease than epi-
nephrine. In one experiment norepinephrine
caused a fall only, although epinephrine caused
a rise. These results show that epinephrine causes
a greater rise in portal pressure than norepi-
nephrine and supports the conclusion of Ahlquist
et al. (J. Pharmacol. & Exper. Therap. 110: 352,
1954) that the reactions of the dog to these 2
sympathomimetic amines are qualitatively similar
but quantitatively different. (Supported by
USAF-SAM Contract AF 19(604)1093 and PHS
Grant #* H-14SOC.)

73. Uneven ventilation of the lungs and the
alveolo-arterial oxygen tension gradient
in normal subjects, breathing air. W. A.
BriscoE* AND ANDRE CouRNAND. Dept. of Medi-
cine, Columbia Univ., New York City.

The distribution of ventilation to 2 or more
groups of alveoli within the lung can be assessed
by washoyt studies with an insoluble indicator gas
and, if the working assumption of homogeneity
of pulmonary perfusion is made, the inequality of
alveolar ventilation-perfusion ratios within the
lung can be defined. Graphic methods have been
developed for dealing with the effects of uneven
ventilation-perfusion ratios on blood gas transfer.
For any measured degree of uneven ventilation
of the lungs it is possible to predict the alveolo-
arterial oxygen tension gradient which would be
caused by uneven ventilation-perfusion ratios if
the lungs were evenly perfused. Eighteen distribu-
tion studies were made in 6 normal subjects using
10% Helium in air as indicator gas in open circuit.
The predicted alveolo-arterial oxygen gradient
averaged 9.2 mm. (range 0-20 mm). This is ap-
proximately equal to the total gradient measured
in such subjects by others. The predicted gradients
due to uneven ventilation-perfusion ratios should

FEDERATION PROCEEDINGS

Volume 16

be less since the total alveolo-arterial oxygen gra-
dient contains other components, particularly
that due to true anatomical shunts by passing the
alveoli. If the predicted gradient equals or ex.
ceeds the observed total gradient the only pos.
sible explanations are either that there has been
error in measurement or else that the less venti-
lated alveoli are underperfused with blood.

74. Ion excretion during acidosis in relation
to hypotheses on urinary acidification,
WituramM A. Bropsky. Dept. of Pediatrics and
Inst. for Med. Research, Univ. of Louisville
School of Medicine, Louisville, Ky.

A current explanation of urinary acidification
includes such tubular processes as secretion of
H* ions, forced exchange of Na* and Ht, and
forced exchange of Kt and H* ions between lumen
and tubule cells. At present this explanation hag
not been established rigorously, and certain ques-
tions are raised by the following observations:
1) a urine pCO, less than that of renal venous
pCO: (Bropsky. Federation Proc., 14: 18, 19565),
2) recent data of S. Solomon on directly measured
p.d.s across renal tubular cells and 3) data re-
ported herein on ion excretion during acidosis.
Dogs were rendered acidotic by loading with
KH2PO., NaH2PO,, H3PO,, or with various buf-
fered phosphate mixtures. After loading with
acid phosphate solutions, increased excretion
rates of Kt, Na*, and phosphate ions concomitant
with an increase in excretion of titratable acid
were observed. After loading with basic phosphate
solutions, increased excretion rates of Kt, Nat,
and phosphate ions concomitant with a constant
or decreased excretion of titratable acid were ob-
served. Clearances of phosphate were higher than
those of potassium during the acidosis of KH2PQ,
loading.

75. Respiratory elastance measured during
single passive inflations and resistance dur-
ing single passive deflations in normal and
partially paralyzed humans. ALFRED W.
Bropy, Patrick 8. O’Hattoran, Harry J.
WANDER AND Everett L. Ro.ey (introduced by
J. RayMonp Jounson). Dept. of Physiology and
Pharmacology, Creighton Med. School and Re-
gional Respiratory Ctr., Creighton Memorial-
St. Joseph’s Hosp., Omaha, Nebr.

Clinically the limits of an individual’s slow
voluntary respiratory effects may be ascertained
satisfactorily by measurement of his respiratory
volumes and the maximal inspiratory and expira-
tory pressures attainable at near end tidal volumes
(H. A. Rahn et al. Am. J. Physiol. 146: 161, 1946).
Elastance and resistance, the dynamic respira-
tory qualities, may be tested rapidly by ‘single
breath’ procedures as follows: Air from a high

els

sec’
jori
pres
the

tabl
the

type
und
duri
phil
lyoy
but

into

intrs
tatu:
from
Aver
herv:
incre

ume 1§

en gra-
ularly
ing the
or ex-
y pos-
s been
venti-
rd.

lation
ation,
cs and
visville

ication
ion of
‘, and
lumen
on has
1 ques-
ations:
venous
1955),
asured
ita re-
idosis.
r with
is buf-
r with
sretion
mitant
le acid
sphate
, Nat,
nstant
2re ob-
r than
HPQ,

luring
e dur-
al and
sp W.
aRY J,
ced by
gy and
nd Re-
morial-

s slow
‘tained
iratory
expira-
olumes
1946).
espira-
‘single
a high

March 1956

pressure source (vacuum cleaner pump) is ad-
mitted into the subject’s mouth at a constant,
measured rate controlled by a high added re-
sistance. If the patient is relaxed and has a con-
stant respiratory resistance, the air pressure in
the mouth will be a constant amount higher than
alveolar pressure. Therefore, the change in mouth
pressure with volume will equal the change in
alveolar pressure with volume and thus is the
elastance. At the end of such an inflation, a pas-
sive deflation may be produced by rapidly stop-
ping the inflating air stream and simultaneously
opening a vent from the mouth to outside air.
Resistance may be calculated from the record of
this deflation (A. W. Brody. Am. J. Physiol. 178:
189, 1954).

16. Early hypotension induced in the rabbit
by whole body x-irradiation. Putturps M.
Brooks AND HERBERT B. GERSTNER (intro-
duced by A. P. Gaaer). Dept. of Radiobiology,
USAF School of Aviation Medicine, Randolph
Air Force Base, Texas.

Two types of experiments were performed. In
the first type, the aortic blood pressure and the
respiratory chest movements were continuously
recorded of 21 rabbits that received 630 r total
body x-irradiation at a dose rate of 10.6 r/min.
and these recordings were compared with those of
15 control animals. All irradiated rabbits showed
4 pronounced initial reaction characterized by
severe hypotension and acceleration of respira-
tory rate. This reaction became noticeable in the
second half of the irradiation period; in the ma-
jority of animals circulatory collapse with a mean
pressure of 50 mm Hg or less developed during
the first postirradiation hour. Thereafter, all
rabbits began to recover from the collapse with
the exception of 4 animals that died. In the second
type of experiments, blood of 16 animals irradiated
under the previous conditions was withdrawn
during the collapse phase and the plasma lyo-
philized. In comparison with control plasma, the
lyophilized irradiated material produced a slight
but consistent fall in blood pressure when injected
into nonirradiated test rabbits.

7. Action of repetitive nerve volleys and of
botulinum toxin on miniature endplate
potentials. V. B. Brooxs (introduced by B.
De.isLE Burns). Dept. of Physiology, Aus-
tralian Natl. Univ., Canberra.

Miniature endplate potentials were recorded
intracellularly from the guinea pig isolated ser-
ratus anterior muscle. Average frequencies range
from 1 to 30/sec., most commonly about 3/sec.
Average amplitudes are about 1 mv. Repetitive
herve stimulation for 20-30 sec. at 200-300/sec.
increases frequencies to 100-150/sec., without

AMERICAN PHYSIOLOGICAL SOCIETY 25

changing amplitudes. The effect decays approx-
imately exponentially to normal values in 3-10
min. and can be reproduced several times on the
same preparation within a few minutes. Smaller
number of stimuli are less effective. The data sug-
gest that the number of ‘packets’ of ACh penetrat-
ing from nerve to muscle is increased without
change of their mean size. Since this process may
be concerned with potentiation of transmission
through partially blocked junctions, this was
tested for paralysis produced by (type A) bo-
tulinum toxin. Excised serratus preparations were
soaked in solutions of 10--10-*. After a ‘latent
period’ of 20-40 min. the frequency of miniature
potentials decreases smoothly to zero, accompa-
nied by a proportional decrease of amplitude of
the e.p.p. However, during the initial 10 min. of
block (approx.), junctional transmission can be
restored by repetitive nerve stimulation, pro-
vided that the frequency of miniature potentials
is raised to levels normal for that junction. There-
after the action of botulinum toxin is irreversible.

78. Influence of Diamox on posthypercapnic
sequelae. E. B. Brown, JR. AND Roya Hay-
DEN.* Dept. of Physiology, Univ. of Minnesota,
Minneapolis.

One of the few effective measures in preventing
the sequelae of rapid reversal of prolonged hyper-
capnia in dogs (cardiac arrhythmias, hypotension,
ventricular fibrillation or cardiac arrest) is that
of gradual rather than abrupt reduction of the
CO: tension of inspired air. The purpose of this
investigation was to determine whether Diamox
would interfere sufficiently with the elimination
of CO: when dogs were quickly changed from
breathing 40% CO:z to 100% Oz to prevent these
serious sequelae. Dogs were allowed to breathe
30% CO+ for 2 hr., followed by 40% CO: for another
2 hr., and were then hyperventilated on 100% O2
with a positive pressure respirator. This procedure
produces ventricular fibrillation and deaths in
80% of untreated dogs. Five dogs were given
Diamox by intravenous injection in a dose of 50
mg/kg b. wt. 5 min. before changing from 40% CO,
to oxygen. None of these dogs showed any cardiac
arrhythmias, or severe hypotension and none died.
In 4 other experiments the rate of rise of arterial
blood pH was determined during the first 10 min.
after switching from 30% CO: to O2 with and with-
out administration of Diamox. Following 50 mg/kg
of Diamox the rate of rise of pH was significantly
slower than it was in the control run. A corre-
spondingly slower fall in pCO2 was observed. This
decreased rate of change of hydrogen ion concen-
tration and pCO: probably accounts for the pro-
tection afforded by Diamox.

26 FEDERATION PROCEEDINGS

79. Lung area from surface tension effects.
Etwyn S. Brown (introduced by D. B. D111).
Chem. Corps Med. Labs., Army Chem. Ctr., Md.
The estimate by Radford of the internal surface

area of the lung from the difference in static pres-

sures of lungs inflated by air and by saline is very
much smaller than anatomic estimates. The as-
sumption that surface tension remained constant
during deflation at a value approximately that of
plasma has been shown to be untenable on several
grounds. Pattle has demonstrated the presence of
material with surface active properties in bubbles
expressed from cut lung tissue. The properties
of similar surface active material in nasal mucus
have been examined. Using the surface tension
data derived from nasal mucus, recalculation of
surface area from Radford’s data and from data
in other species yields values for lung surface
area more in accord with anatomic estimates.
From these values the relative size of units par-
ticipating in lung expansion and the relative num-
ber of such units have been calculated. Lung
volume, surface area, and number of units were
related to body weight of various species. The size
of the lung unit as calculated from these data ap-
peared to be nearly independent of body weight
and was in accord with histologic evidence. Extra-
polation of these data to a body weight of 70 kg
gave the resting lung volume as 2.5 1., the surface
area as 60 m?, the number of expansible units as
500 million, and their mean diameter as 0.25 mm.

80. Assay of exogenous TSH by a two-day
assay using normal rats. J. H. U. Brown.
Dept. of Physiology, Emory Univ., Emory Uni-
versity, Ga.

A method has been developed for the assay of
TSH in normal animals. Littermate males of about
100 gm in weight were given a subcutaneous in-
jection of 1 ml of 5% Nal. Eighteen hours later,
the animals were divided into groups of 4 animals;
1 group sérved as a control and the other groups
received TSH in aqueous solution (0.5 ml) in the
desired dosages. Six hours following the TSH in-
jection all animals were given an injection of 3
uc of I'*! (0.5 ml). Eighteen hours after the iodine
injection, the animals were killed, the thyroids
were removed, dissolved in 1 ml of 10% NaOH,
and counted in a well-type scintillation counter.
Appropriate dilutions were made if necessary.
With this method normal animals had an uptake
of 6500 counts/min. above background; animals
given Nal alone had an average count of 300
counts/min.; with 0.013 vu of TSH the count was
560 counts/min.; with 0.065 u of TSH the count
was 890 counts/min.; and with 0.26 vu the count
was 1400 counts/min. In any given experiment the
standard deviation averages about 10% of the
count but the dose response curve varies from

Volume 1§

experiment to experiment due to variations in
iodine dosage, etc. Unknowns can be readily ag.
sayed by bracketing the unknown with known
standards. Normal rat plasma has been assayed
by giving 1 ml of fresh plasma under the outlined
conditions and averaged 1.5 v/100 ml plasma,
Plasma from thyroidectomized rats averaged 35
u of TSH/100 ml of plasma. The method is suitable
as a quick screening method and is not designed to
replace methods with a greater degree of accuracy,
(Supported by the Damon Runyon Memorial
Fund and PHS Grant A-987.)

81. Metabolism of cortisone in tissue slices,
J. H. U. Brown anp ATALANTE ANASON. Dept,
of Physiology, Emory Univ., Emory University,
Ga.

Slices of kidney and liver were incubated in
Kreb’s solution containing varying amounts of
cortisone. After incubation the slices were re-
moved, washed free of media, homogenized and
extracted with CHCl; after precipitation of pro-
teins with TCA. The media was also extracted with
CHCl;. Both fractions were analyzed for steroids
by the methods of Porter-Silber and Gornall, and
by measurement of the extinction at 240 mu. The
results were expressed as gain in steroid in the
experimental slices over the control slices to which
no steroid was added and as loss of steroid from
the media as compared to the loss from the media
when dead or frozen slices were used. Recovery
of added steroid was 92-103% complete. Little
or no steroid was detected in slices or media to
which no steroid was added. Tissues of animals
pretreated with cortisone, of hypophysectomized
animals and/or animals treated with triiodothyro-
nine (T.I.T.) were compared to normal control
slices. Hypophysectomized animals were able to
utilize less cortisone from the media and cortisone-
treated animals were able to utilize more cortisone
from the media than were normal controls. Ani-
mais treated with T.I.T. appeared able to handle
steroid more effectively than normals. There was
a suggestion that the slices metabolize cortisone
more effectively in the cortisone treated animal.
The metabolism of kidney is much less than that
of liver. Values for the Porter-Silber and Gornall
reactions parallel each other closely but the values
for E-240’s do not check as closely. All slices
showed a high correlation in metabolism with the
weight of tissue, temperature of incubation, time
of incubation and amount of steroid added to the
slices. (Supported by grants from the Damon
Runyon Memorial Fund and the PHS, Grant
C-2339)

82. Concurrent effects of x-irradiation on
peripheral blood and peritoneal fluid cell
populations in the rat. F. G. Bucu,* H. A.

fre
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if

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ons in
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itlined
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uracy,
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, Dept.
ersity,

ted in
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March 1956

CHARIPPER AND A. EpELMANN. Biology Depts.,
New York Univ., and Brookhaven Natl. Lab.,
Upton, N. Y.

Comparisons of peripheral blood and _peri-
toneal fluid cell populations 2 and 6 days following
whole-body x-irradiation (750-1000 r) were made
in untreated and pollen-sensitized rats. X-irradi-
ation of untreated rats resulted in a decrease in
total peritoneal fluid cell population which was
less severe than the reduction in the number of
peripheral blood cells. The decrease in the number
of peritoneal fluid eosinophils was as acute as that
found in the blood. Mast cells in peritoneal fluid
decreased in number at a more gradual rate than
did the total cell population. In pollen-sensitized
rats the reduction in cell population 2 days after
x-irradiation was not significant even though there
was a marked decrease in the total number of
blood cells. At 6 days after exposure the reduction
was significant in both fluids, though less marked
in peritoneal fluid. Eosinophils dropped markedly
in number in both fluids. There was no change in
the number of mast cells in the x-rayed pollen-
sensitized rat. Since the effect of x-irradiation on
the eosinophils is as marked in pollen-sensitized
rats as in untreated rats, even though the numbers
of blood and peritoneal fluid eosinophils increased
in non-x-rayed pollen-sensitized rats, it seems that
the eosinophil is extremely radiosensitive and is
affected independently of the other cells.

83. Implantation in ovariectomized armadil-
los. G. D. Bucuanan,* A. C. ENDERS* AND
Roy V. TatmaGe. Rice Inst., Houston, Texas.
The effect which ovariectomy performed in the

interval between ovulation and implantation has

on the fertilized ovum has scarcely been investi-
gated. Animals exhibiting delayed implantation
provide an extended time period in which such
experiments can be performed and difficulties such
as possible effects of ovariectomy on tubal passage
can be obviated. The effect of ovariectomy on the
free blastocyst in the armadillo has been studied.

If bilateral ovariectomy is performed mid-way

in the 4-month delay period, implantation occurs

30-34 days later and is indistinguishable from

normal implantation. One animal was maintained

94 days after ovariectomy and contained 4 normal

fetuses of the size comparable to fetuses at this

stage in normal pregnancy. In animals ovariec-

tomized toward the end of the delay period, im-

plantation occurred sooner and at the time when

normal implantation was expected. However,
resorption or abortion followed shortly thereafter.

Removal of the corpus luteum by unilateral

ovariectomy at this latter period for the most

part caused blastocyst loss instead of implanta-
tion. It is possible that ovariectomy in armadillos,
if done sufficiently early in the delay period,

AMERICAN PHYSIOLOGICAL SOCIETY 27

produces enhanced hypophyseal activity, which in
turn stimulates some nonovarian tissue (adrenal?
or uterus?) to produce estrogen and progesterone.
The titers realized correspond to these normally
produced at the end of the delay period and
nidation occurs. Ovariectomy performed late in
the delay period cannot prevent normal implanta-
tion but the alternate steroid-producing tissue is
then insufficient to maintain pregnancy. Corpus
luteum removal at this time appears to upset the
uterine physiology such that the blastocyst is
destroyed.

84. Behavioral and action potential responses
to stimulation of subcortical structures in
the unanesthetized cat. N. A. BucHwaLp
AND F. R. Ervin (introduced by L. M. N.
Bacn). Dept. of Physiology, Southeast Louisiana
Hosp. and Dept. of Anatomy, Tulane Univ., New
Orleans.

Teflon-insulated silver electrodes (total diam-
eter about 7 mils, recording tip diameter less than
5 mils) were inserted stereotactically in bilaterally
symmetrical subcortical locations in cats. Stimu-
lations of unanesthetized animals were carried out
at a number of points presumed to be related to
the amygdaloid complex in one experimental
series and to the globus pallidus in another series.
For each electrode site systematic stimulation
employing a wide range of voltages, frequencies
and pulse durations were carried out. Marked
variations in behavioral responses from given loci
were observed with changes in parameters of
stimulation. Many responses similar to those re-
ported by Kaada et al. (Neurol. 4: 148, 1954) in
stimulation of the amygdaloid were, however,
seen. Bipolar, single shock stimuli of short dura-
tion (0.01-0.1 msec.) were applied to the globus
pallidus, amygdala, caudate nucleus, mesen-
cephalic tegmentum and other sites. Recordings
from these and other subcortical structures
showed consistently repeatable electrical re-
sponses, although with few exceptions behavioral
responses were not observed with brief single
shocks. Responses of short latency (one to 5
msec.) were noted following the 0.01-0.1 msec.
shocks. Many of the early responses were ob-
scured by the stimulus artifact when longer dura-
tion (1-5 msec.) stimuli were delivered. Histo-
logical examination of electrode locations is being
carried out.

85. Skin water intake in normal persons and
during menstrual cycle, pregnancy and
cardiac edema, and concentration of sweat.
Konrap J. K. Buettner. Depts. of Meteorology
and of Physiology, Univ. of Washington, Seattle.
If the water vapor pressure on a small non-

sweating skin area exceeds a critical figure, vapor

flows through the skin into the body. For vapor

28 FEDERATION PROCEEDINGS

pressures below this critical or ‘neutral’ point
this flow is reversed. The same transfer results
when osmotic solutions cover the skin. The
neutral humidity of vapor applied corresponds to
the neutral osmolarity of the solution. Both data
are expressed as relative humidities at skin tem-
perature. The neutral relative humidity (NRH)
for the arm of normal persons is found as 85%.
This corresponds to air in contact with saturated
KCl. During menstrual cycle, NRH as low as 60%
are on record. Many pregnant and cardiac patients
show low NRH, especially in severe edema,
toxemia of pregnancy, severe angina, and near
parturition. The observed or net flow is the sum
or difference of the above-described diffusion and
sweat. Diffusion flow may exceed 100 gm/m?/hr.
either way. All sweat concentration data have,
therefore, to be retested since no solutes seem to
accompany the diffusion flow. The so-called
insensible perspiration is a sum of at least two
components: diffusion and the continual sweating
in palms, axilla, peritoneal region, etc. Insensible
perspiration is an inappropriate term describing
two separate processes.

86. Maintenance of arterial pressure and
cardiac output in the hypothermic rat.
R. W. Butuarp (introduced by E. F. ADoupH).
Dept. of Physiology, Univ. of Rochester, Rochester,
N.Y.

The unanesthetized hypothermic rat maintains

a mean arterial blood pressure of over 100 mm Hg

until cooled below 18°C. A study of other circula-

tory factors might reveal the mechanisms in-
volved in maintaining it. Unanesthetized rats
were cooled in water to various temperatures
between 36°C and 14°C. Cardiac output, mean
arterial blood pressure, pulse pressure, venous
blood pressure, heart rate and blood viscosity
were measured at selected temperatures. Cardiac
outputs, as estimated by a modified dye dilution
method, decreased from a mean of 210 ml/kg
min. (S.D. = +45) at 36°C to 35 ml/kg min.
(+5) at 16°C. Heart rates decreased from a mean
of 424/min. at 36°C to 51/min. at 16°C. The only
consistent change in pulse pressures was a de-
crease occurring as the lethal temperature of 15°C
was approached. The arterial pressure remained
high while the cardiac output was decreasing,
hence the calculated total peripheral resistance
was increased in cold animals. The viscosity of the
blood, as measured in a capillary tube, increased
with lowered body temperature. This increase
accounted for most of the increase of the total
peripheral resistance. However, at all tempera-
tures transient variations in mean arterial pres-
sure and pulse pressure occurred which could be
explained only by vasomotor activity. Therefore,
the maintenance of arterial pressure despite the

Volume 1§

lowered cardiac output of the hypothermic rat
depends upon increased peripheral resistance,
most of which is due to the increase of blood
viscosity and little to vasoconstriction.

87. Effect of crystalline vitamin B,2 and of a
crystalline grasshopper pigment (GHP) on
experimental anemia in mice. L. E. Buragss,*
S. S. Crark* anp D. T. Roxre. Physiology
Dept., Meharry Med. College, Nashville, Tenn.
The isolation of a reddish-brown crystalline

substance from the developing egg of the grass-

hopper has been reported (Arch. Biochem. 20: 347,

1949). This reddish-brown substance exhibits

infrared absorption spectra similar in character to,

but not identical with, the spectra of vitamin Bj».

The present investigation was conducted to

determine how the reddish-brown crystalline

grasshopper pigment compares with vitamin By,
in stimulating red blood cell production in Swiss
mice rendered anemic by the administration of
phenylhydrazine hydrochloride. The _ experi-
mental procedure used in this investigation was
essentially the same as the technique used by

Vijayaraghaven et al. (Proc. Exper. Biol. & Med.

75: 754, 1950). For a period of 10 days, 1 group of

anemic mice was given daily intraperitoneal in-

jections of vitamin B,2 while another group was
given injections of the grasshopper pigment.

The results of these studies indicate that the

reddish-brown substance can replace vitamin By;

in stimulating red cell production in mice rendered
anemic by phenylhydrazine hydrochloride. In
these mice, the stimulation of red blood cell
production by the reddish-brown compound was
slightly greater than that of vitamin Bis. The
number of red blood cells per unit volume, but
not hemoglobin concentration, was increased by
the reddish-brown grasshopper pigment. The
increase in red blood cells was roughly propor-
tional to the amount of reddish-brown pigment
administered. This pattern was most obvious
during the first 4 days of severe anemia. (Aided
by a grant from the Natl. Insts. of Health, PHS.)

88. Variation in resting oxygen consumption
throughout the day. E. R. BuskIRK AND
P. F. Iampretro (introduced by Austin HEn-
SCHEL). Quartermaster Research & Develop. Ctr.,
Natick, Mass.

Diurnal variation in resting metabolism was
measured during several large field studies to
ascertain reasonable ‘base line’ values for oxygen
consumption at any given hour and to ascertain
the impact of environment on resting metabolism.
Eight men were studied for at least 10 days in each
of 4 climates. Weather conditions ranged from
hot-dry at Yuma, Arizona to cold-dry at Fort
Churchill, Manitoba Canada. The subjects sub-

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March 1956

sisted on standardized rations during each experi-
ment. Oxygen consumption was routinely meas-
ured at 8 A.M. (pre-breakfast), noon (pre-lunch),
4p.M. (pre-supper) and 8 p.m. hr. each day after
30 min. rest in a supine position. Oxygen con-
sumption at 8 a.m. was significantly lower than
that at any other hour. The noon and 4 p.m.
yalues were not different from each other, but the
§p.M. value was significantly higher than that
at any other hour in each environment. A major
portion of the elevation in metabolism during the
day was associated with ‘Specific Dynamic Ac-
tion.’ Thus, when men fasted and exercised
moderately or fasted with no exercise the daily
elevation in metabolism was present but was
significantly less than when food was given. Prior
moderate exercise had little measurable effect on
the resting oxygen consumption. The same pattern
of results was observed in each environment. It is
concluded that the diurnal pattern of oxygen
consumption is little affected by environment
within the range studied.

§. Basic physiologic studies on normal
children following mild chlorpromazine
sedation. J. D. Buxspaum,* L. M. Provutrt,*
F. Erticn* anp R. H. Oster. Depts. of Phys-
tology, and Pedodontics, School of Dentistry,
Univ. of Maryland, Baltimore.

Fifteen children ranging from 5-12 years sched-
wed for pedondontic treatment were given from
10-20 mg. of chlorpromazine to alleviate the
usual apprehensive approach to dental pro-
cedures. This use of a tranquillizer drug in ac-
cordance with a hypothesis of cortical modulation
of the pain and fear response requires quantitative
physiologic evaluation. The following physiologic
procedures were carried out prior to therapy and

then duplicated under therapy: cold pressor test -

(Hines AND Brown, Ann. Int. Med. 7: 209, 1933),
qualitative sweat responses (RANDALL, J. Clin.
Investigation 25: 761, 1946), pulse rate respiratory
volumes and complete electroencephalograms.
The drug was administered on the previous even-
ing and the dose repeated 1-2 hr. before testing.
The slopes of the cold pressor graphs showed no
difference in pre- and post-medication data, but
the response was delayed 30 sec. after sedation.
The patch sweat test was inconclusive: 8 of 15
subjects showed post-medication increase. The
pulse rate showed no consistent change. The
respiratory volumes decreased an average of 5.3
l/min. after medication, 14 of the 15 subjects
showing decreases of 0.5-12.9 1/min. EEG’s were
tarefully analyzed including per cent time oc-
currence of alpha, beta and delta waves and
hyperventilation effects. The alpha and delta
waves showed no significant difference after
medication. Beta waves were counted in frontal,

AMERICAN PHYSIOLOGICAL SOCIETY 29

parietal, and occipital regions unilaterally: the
frontal areas showed a statistically significant
drop in per cent time beta after medication.

90. Ionic relationships between muscle cells
and their surrounding connective tissue.
Tuomas B. CatHoon (introduced by T. G.
BERNTHAL). Dept. of Physiology, Med. College of
South Carolina, Charleston.

Studies have been made in an attempt to deter-
mine directly the influence of connective tissue
on the movement of ions between an immersion
fluid and ‘whole muscles.’ Rat gastrocnemius
muscles were immersed in a Ringer’s solution
whose sodium and potassium contents were
determined flame-photometrically before and
after immersion. After an immersion period of
2 hr. the water content of the muscles was deter-
mined. From these data it was found that these
‘whole muscles’ gained 18% weight, 0.8 mg. of
sodium/gm/hr. and 92 mg. of water/gm/hr.,
while they lost 0.2 mg. of potassium/gm/hr.
Similar procedures were carried out using muscle
fascia and subcutaneous connective tissue. These
were found to have gained 160% weight, 6.0 mg.
of sodium/gm/hr., 792 mg of water/gm/hr. and °
0.05 mg of potassium/gm/hr. Thus, knowing the
amount of connective tissue in a ‘whole muscle,’
it was possible to determine to what extent the
connective tissue, contained within the muscle,
accounts for the shifts of sodium, potassium and
water into or out of the ‘whole muscle.’ These
procedures were repeated in the presence of
hyaluronidase to determine if disturbance of the
mucopolysaccharide of the connective tissue
modifies this picture.

91. Quantum detection in biological systems,
with particular reference to olfaction.
Berry CaMpBELL. Dept. of Anatomy, Univ. of
Minnesota, Minneapolis.

Detection in the most sensitive sense organs
demands the production of a ‘mega-event,’ the
generation of a nerve impulse, from a ‘micro-
event,’ involving a small number of quanta. In
the case of the retinal rod cells, the ultimate effi-
ciency is reached where one photon suffices for a
signal. The remarkable sensitivity of the ol-
factory hair cell poses a similar question. A pre-
liminary hypothesis has been erected which con-
siders the particular problem of the olfactory
hair. This detector hair is birefringent and highly
organized. It is proposed that it serves as a path of
conduction of the standing potential of the ol-
factory cell and that the absorption of an aro-
matic molecule is the mechanism for a sudden
alteration of conductance. This results in a
prompt readjustment of the cell dipole and of the
leakage of current from the first part of the axon—

the stimulus for depolarization. It was seen that a
carotene containing repetitive crystal-like struc-
ture, comparable to the outer segment of the
retinal rod would be a likely mechanism. The
alternate double bonding of the carotenoid chains
would serve as a quasi-metallic conductance and
there would be provided the active termini for
substitution. A search for such a carotenoid con-
tent of the olfactory hairs was then made. In the
olfactory hairs of the frog, ultraviolet microscopy
revealed a light-green fluorescing substance fitting
the demands of carotenoid. Extension of these
observations to other detecting hairs, as in the
inner ear, is being made. The hypothesis that
detection at or near the single quantum level is
possible in biological systems where quasi-metallic
conductance may be modulated by signals ap-
proaching the ultimate threshold is suggested.

92. Radioactive phosphorus uptake by hu-
man platelets. Epmunp W. CAMPBELL AND
Wa.Ter J. SMALL (introduced by M.S. RaBen).
Blood Research Lab., New England Ctr. Hosp.,
Boston, Mass.

The uptake of P32 by human platelets has been
applied to the study of their viability, metabolism
and function. Blood was collected in several
anticoagulants using ‘Silicone technique.’ Plate-
let-rich plasma obtained by centrifugation at 1500
rpm for 15 min. at 4°C was incubated with Ps.
Radioactivity of platelet-rich plasma and washed
platelets was measured by a G-M tube. Direct
platelet counts were done on all samples. Total
radioactivity, incorporated by the platelets, was
directly proportional to the total number of
platelets per sample and the amount of Ps2 added
but inversely proportional to the sample volume.
Each of these 3 parameters was varied in turn and
the proportionality constant was found to be in
agreement. The effects of variation of controlled
conditions, including storage, px, centrifugation,
time and temperature of incubation, on this
constant were evaluated using freshly-collected,
normal platelets. Platelets collected from abnormal
individuals were studied. Correlative study of
platelet survival and effects in blood coagulation
was done simultaneously. The value of the con-
stant was reproducible in the same normal indi-
vidual and the dispersion of values obtained in
many normal individuals was well within the
range of experimental variation. The variation
of controlled conditions caused striking differ-
ences in the values obtained. The values obtained
in abnormals differed markedly from the normal
range under carefully controlled conditions. A
technique which promises to be useful in evaluat-
ing human platelet viability, function and ab-
normality has been described. (Supported by a
grant from the Atomic Energy Commission.)

FEDERATION PROCEEDINGS

Volume if

93. Estimation of pulmonary tissue volume
and pulmonary capillary blood flow by
analysis of respired soluble inert gases,
L. Canper* and R. E. Forster. Dept. of
Physiology and Pharmacology, Grad. School of
Medicine, Univ. of Pennsylvania, Philadelphia.
Previous work on the rate of disappearance of a

soluble inert gas from the lungs has been handi-

capped by the difficulty of predicting the initial
gas concentration in a given alveolar gas sample
owing to uneven distribution of inspired gas. By
including a relatively insoluble inert gas (helium)
in the inspired gas mixture, one can calculate the
initial (prior to uptake by blood and tissue)
mixed concentration of the soluble inert gas in the
alveolar sample from the helium dilution. In this
manner we have measured the disappearance of
five gases relative to helium over different periods
of breath holding up to 50 sec. in two healthy
subjects. The gases studied were sulfur hexa-
fluoride, nitrous oxide, acetylene, diethyl ether,
and acetone, the reported Ostwald coefficients of
which for blood range respectively from 0.001 to
approximately 300. The concentration of any one
of these five gases was measured continuously at
the mouth by a mass spectrometer; end expiratory
helium concentration was measured almost
simultaneously. All of the soluble gases show an
abrupt fall in alveolar concentration relative to
helium within the first 2 sec. of breath holding,
and thereafter (except for acetone which is a
special case) disappear at a much slower rate
which depends upon their solubilities in blood and
pulmonary capillary blood flow. The initial
abrupt fall in concentration apparently represents
an extremely rapid equilibration of the soluble
gas with a pulmonary tissue volume of approxi-
mately 500 ml.

94. Incorporation of uracil-2-C" into nucleic
acids of regenerating liver. A. CANTAROW,
T. L. Writrams* anp K. E. Pascuxts. Div. of
Endocrine and Cancer Research, Jefferson Med.
College, Philadelphia, Pa.

We have reported that uracil is incorporated in
nucleic acids of normal rat liver in vitro, and,
in vivo, in nucleic acids of preneoplastic and
neoplastic livers of rats receiving 2-acetamino-
fluorene. This phenomenon was previously not
believed to occur in mammalian tissues. It has
since been reported to occur in intestinal epi-
thelium. The present experiments demonstrate
incorporation of uracil-2-C“ and of thiouracil-2-
C™ in nucleic acids of regenerating liver. No
incorporation was demonstrable in the livers of
thyroidectomized normal rats and diminished
incorporation occurred in regenerating liver in
thyroidectomized (partially hepatectomized) ani-
mals. Data will be reported also on the effect of

med
of is

and
disa
on f
Qos
resp
pyrt
iner
it es
inte:
two
oxys
herv

Qos i
Part
or xt

lume 15

volume
low by
| Sases,
Dept. of
chool of
elphia.
ince of a
1 handi-
e initial
; Sample
gas. By
helium)
late the
tissue)
a8 in the
. In this
rance of
periods
healthy
ir hexa-
‘l ether,
ients of
0.001 to
any one
ously at
piratory
almost
show an
ative to
holding,
ch is a
yer rate
ood and
initial
presents
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nucleic
'TAROW,

Div. of
m Med.

rated in
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It has
al epi-
mnstrate
racil-2-
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inished
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March 1956

thiouracil ep incorporation of uracil in AAF-
treated rats.

9. Synergistic action on growth of Neuro-
spora crassa of pyrimidines following
irradiation and _ reconstitution. ATTILIO
CANZANELLI, RHEA SossEN* AND Davip Rap-
porT. Dept. of Physiology, Tufts Univ. School of
Medicine, Boston, Mass.

Upon irradiation of uridine with ultraviolet
light there is a progressive loss of the selective
ultraviolet absorption spectrum with a con-
comitant loss of the capacity of the irradiated
substance to support growth in a pyrimidine-
requiring mutant of Neurospora crassa. We have
shown that in the irradiated product there has
been a hydration of the conjugated bond, which
can be reconstituted by boiling, acidification or
alkalinization. The reconstituted material may
consist of uridine and uracil, alone or together,
plus a small amount of other unidentified material,
as determined by paper and ion-exchange chro-
matography and spectrographic data. The growth
of the mutant on the reconstituted material is
greater than can be accounted for by uridine
or uracil alone. Investigation of this showed that
it was apparently due to a synergism between the
growth-promoting capacities of the 2 substances.
This has been demonstrated for pure uridine and
uracil. The evidence indicates that the presence
of uridine in the nutrient medium facilitates the
utilization of uracil by the mutant.

%. Alteration in mammalian nerve pyruvate
metabolism by anesthetic gases and barbi-
turates. FRANK G. CARPENTER (introduced by
J. F. MacLeop). Dept. of Physiology, Cornell
Univ. Med. College, New York City.

In 28 experiments in which Na phenobarbital
(10-15 m/l) was present in a buffered Ringer
medium of pH 7.4, reversible conduction blockade
of isolated rat sciatic nerves was accompanied by
a60-70% reduction in their resting oxygen uptake
and a 50-60% inhibition in the rate of pyruvate
disappearance. 5-7 m/l is without visible effect
on fiber conduction yet the resting Qo. and the
Qosruvat» are inhibited 30-40% and 20-30%
respectively. Although the addition of 10 mm/1 Na
pyruvate to isolated peripheral nerve produces no
increase in oxygen uptake (nonsubstrate limited)
it can be shown by chemical analysis that this
intermediate decreases in a linear fashion over a
two hour period in the presence of 1 or 2 atm. of
oxygen. Also when the glycolytic pathways in the
nerves are partially abolished by 20 mm/] NaF or
1 mm/l Na iodoacetate the usual reduction in
Qo2 is prevented if 10 mm/1 Na pyruvate is present.
Partial pressures of cyclopropane, nitrous oxide
or xenon sufficient to produce reversible blockade

—_
a8 the

AMERICAN PHYSIOLOGICAL SOCIETY 31

of impulse transmission has been found to be
without demonstable effect on either the rate of
pyruvate disappearance or the oxygen utilization
by peripheral nerves measured at 2.0, 10 and 13
atm., respectively.

97. Dietary fatty acids and cholesterol excre-
tion in the rat. K. K. Carrouu anp R. L.
NoBLE (introduced by J. B. Coxutp). Collip
Med. Research Lab., London, Ontario, Canada.
Studies in this laboratory have shown that feed-

ing the mono-ethenoid fatty acids erucic acid (Cz)

and nervonic acid (C24) to rats caused deposition
of cholesterol in the adrenal cortex (CARROLL,

J. Biol. Chem. 200: 287, 1952). In further studies

where 10-15% by weight of these fatty acids was

added as the sole fat to a sterol-free synthetic
diet, the concentration of cholesterol in the feces
was increased by 2 to 4-fold, without apparent
depletion of the total body cholesterol. The fecal
cholesterol was measured both colorimetrically
and by isolating and weighing the digitonide and
was further purified by chromatography on
alumina and identified by melting point and
infrared spectrum. LEicosenoic acid, the C2
analogue, also increased the fecal cholesterol con-
centration although it had little effect on adrenal
cholesterol. Oleic acid and various even-membered
saturated fatty acids from C, to Cx failed to in-
crease the concentration of cholesterol in the feces
appreciably. However, the long-chain saturated
acids were poorly absorbed and so increased both
the weight of feces and the total amount of cho-
lesterol excreted. This effect was also observed
with erucic and nervonic acids in addition to the

increase in fecal cholesterol concentration. A

preparation of spinal cord cerebrosides fed at the

25% level failed to affect adrenal cholesterol, and

although there was some increase in fecal cho-

lesterol excretion the results were complicated by
poor absorption and by the presence of small
amounts of cholesterol in the preparation.

98. Transfusions of blood from patients with
hemophilia ‘A’ into normal and thrombo-
cytopenic recipients. Ropert T. CARROLL,*
Ruta R. Hoisurn,* L. Barrp* anp L. M.
Tocantins. Charlotte Drake Cardeza Fndn.,
Jefferson Med. College, Philadelphia, Pa.

Blood obtained with silicone-coated syringes
from fasting subjects with various grades (mild =
grade I to severe = grade IV) of hemophilia ‘A’
was injected slowly, immediately after collection,
without added anticoagulants, into normal and
thrombocytopenic individuals of compatible blood
groups. When 300 ml of blood from a hemophiliac,
grade IV, was injected into a normal person, clot-
ting time of venous blood in glass tubes was pro-
longed from 8 min. (pretransfusion) to 138 min.

32 FEDERATION PROCEEDINGS

(posttransfusion) and in silicone-coated tubes
from 23 to 268 min., respectively; the same blood
given to a thrombocytopenic person prolonged the
clotting time of the recipient’s blood in glass from
144 to 473 min. and in silicone from 84 to 540+
min. When 300 ml of blood from a grade IT hemo-
philiac was injected into a normal individual, the
clot delaying effect was negligible, but when
injected into a thrombocytopenic patient, the
clotting time in glass was delayed from 18 to 29
min. and in silicone from 48 to 96 min. Grade I
hemophilic blood injected into either normal
or thrombocytopenic recipients produced no
significant change. The effect induced by trans-
fusion of grade IV hemophilic blood lasted 5 days
but only 2 days when grade IJ hemophilic blood
was transfused. Prolongations of coagulation from
addition of the hemophilic blood to normal blood
in vitro are not as striking as when the mixtures
are made in vivo. Plasma of hyperglobulinemic
patients resists the clot delaying effect of hemo-
philic plasma.

99. Hemodynamic determinants of coronary
flow and myocardial oxygen consumption.
R. B. Cass, 8. J. Sarnorr, E. BRauNwALD,
W.N. Srarnspy anp Z. Taytor (introduced by
J. L. WuttrenBERGER). Lab. of Cardiovascular
Hemodynamics, Natl. Heart Inst., Bethesda, Md.
Using a preparation described elsewhere

(BRAUNWALD et al. Federation Proc., this volume)

it was possible to maintain mean aortic pressure,

cardiac output and heart rate constant, or to
independently vary these parameters over wide
ranges. The following calculations were made:
external left ventricular work = mean aortic
pressure X cardiac output; O2 consumption =
left coronary artery flow X A-V O, difference;
efficiency = work/O2 consumption. The following
was observed: a) there was no straightforward
relationship between work per se and myocardial

Oz consumption. 6) When mean aortic pressure

was progressively elevated (50-200 mm Hg), but

stroke volume and heart rate held constant, O,

consumption increased almost proportionately

with work; there was little change in efficiency.

c) When stroke volume was progressively in-

creased (cardiac output from 1 to 5 1/min.) but

mean aortic pressure and heart rate held constant,
only a slight increase in O2 consumption was ob-
served; a several-fold increase in efficiency oc-
curred. d) When heart rate was increased, but
mean aortic pressure and “ardiac output were held
constant, O2 consumption rose; the efficiency was
thereby decreased. e) Coronary A-V difference
remained remarkably constant over wide varia-
tions in hemodynamic performance in any given
dog at the same hematocrit. It appears from these
data that the rate of oxygen consumption is

Volume if

largely determined by the tension which the
myocardial fiber develops and the frequency with
which it does so, rather than the amount it short-
ens.

100. Resuscitation in the newborn. 8. Cassiy
(introduced by C. E. Hau). Dept. of Physiology,
Univ. of Texas Med. Branch, Galveston.

An investigation of the process of anoxic death
in newborn pups, rabbits and kittens, all less than
24 hours old, was made. Simultaneous measure-
ments of respiration, heart rate and blood preg-
sure were taken. The respiratory rates were seen
to decline slowly, the last breath occurring often
as long as 40 min. after the induction of anoxia,
This preceded plus failure by some 2-60 min. The
heart rate and systolic blood pressure decreased,
slowly in some animals and very abruptly in
others, as anoxia progressed, e.g. the time of
circulatory failure ranged from 30-150 min. The
diastolic blood pressure undergoes an early, fairly
rapid decrease; it then tends to taper off slowly to
a minimum. These extremes in response have been
correlated with variation in the body temperature,
hypothermia greatly prolonging the breathing and
the onset of circulatory failure. Attempts to
resuscitate were made at various points in the
decline of blood pressure. They were successful
even when pulses, as recorded from the carotid
artery, had ceased (electrocardiograms still
present) for about 3 min. Circulatory recovery,
once initiated, was a rather rapid process, the
blood pressure being restored in about 3 min.
However, in many cases, in spite of circulatory
recovery, respiration never did return. When
respiratory recovery did occur, it was slow, breath-
ing being reinitiated after about 13 min. of arti-
ficial respiration.

101. Metabolism of pyruvate-2-C to CO, and
glycogen by isolated perfused cardiac and
skeletal muscle. H. MEap CAvertT AND RvtTH
B. Boyp.* Physiology Dept., Univ. of Minnesota,
Minneapolis.

Sodium pyruvate-2-C' was administered at
levels of 0.5-6 m/l. to the isolated, blood-per-
fused dog heart or gastrocnemius muscle, the
latter either at rest or repetitively contacting
twice per second. Respiratory CO: collected
quantitatively throughout 2-4 hr. of perfusion
contained CO, increasing in percentage with
time or with pyruvate concentration. At com-
parable blood pyruvate concentrations con-
tracting skeletal muscle derived a considerably
greater portion of its respiratory CO, from in-
jected pyruvate-2-C™“ than did resting muscle
under similar conditions (blood glucose 60-100
mg %, 3.2 units of insulin). Values for isolated
hearts slightly exceeded those for stimulated

ditic
a gi
Can:
103.

lume 16

ch the
cy with
t short-

Cassin
siology,

c death
ss than
easure-
d pres-
re seen
g often
anoxia,
in. The
reased,
tly in
ime of
n. The
, fairly
owly to
ve been
rature,
ing and
pts to
in the
‘cessful
carotid
s still
covery,
ss, the
3 min.
ulatory

When
breath-
of arti-

O, and
ac and
» Ruta
nesola,

red at
od-per-
le, the
tacting
llected
rfusion
e with
t com-
; con-
lerably
om in-
muscle
60-100
solated
\ulated

March 1956

gastrocnemius. For example, at 1.7 ma of pyruvate-
9044/1. the percentages of CO, derived from the
administered compound were 2% for resting
gastrocnemius, 6% for contracting gastrocnemius
and 8% for heart. Percentages of injected pyruvate
recovered as CO: were 5, 49 and 25%, respectively.
Glycogen isolated from resting muscle was sig-
nificantly radioactive, but in contracting gastroc-
nemius or heart less than 0.2% of glycogen
carbon was derived from administered pyruvate
carbon. Complete carbon degradation of glucose
fom labeled muscle glycogen revealed that
greater than 90% of the glycogen radioactivity
resided in carbons 2 and 5. This isotopic pattern
suggests that reversal of the Embden-Myerhof
sequence of reactions is the predominant pathway
for glycogen synthesis from pyruvate in isolated
muscle.

102. Effects of growth hormone in hypophy-
sectomized and normal dogs. L. CHarkor,*
R. ZeMEL,* G. A. WRENSHALL,* J. MARKOWITZ
AND J. CAMPBELL. Dept. of Physiology and the
Banting and Best Dept. of Med. Research, Univ.
of Toronton, Toronto, Canada.

The effects of growth hormone (Connaught
Med. Research Labs.), administered subcuta-
neously for 5 days, were compared in 4 normal and
4 hypophysectomized dogs in good condition
(45-80 days postoperative) under the same con-
ditions of environment, diet (horse meat) and
dosage (1 mg/kg/day). The control dogs, 2 normal
and 2 hypophysectomized, were given injections of
saline. The growth hormone increased the body
weight, the erythrocyte sedimentation rate and
the volume per cent of plasma in the intact and
operated dogs; produced diabetes (hyperglycemia,
glycosuria, polyuria and ketonuria) in the intact
dogs and increased blood sugar, but not to diabetic
levels, in the hypophysectomized dogs. It in-
creased the weights (per kg. of body weight) and
the total lipid contents of the liver, kidney and
heart, but to a greater extent in the intact than
in the hypophysectomized dogs. In agreement
with the findings of Essex, Taylor and Wrenshall,
the insulin extractable from the pancreas was
higher in intact than in hypophysectomized dogs
(8.1 vs. 5.1 units/kg of body weight). The ex-
tractable insulin of the pancreas was reduced by
the growth hormone injections much more in the
intact than in the hypophysectomized dogs (to
0.52 and 3.8 units/kg respectively). The growth
hormone injections appeared to improve the con-
dition of the hypophysectomized dogs. (Aided by
4 grant from the Natl. Research Council of
Canada.)

108. Reversible frequency-selective reduction

by cold of round window potentials. ALFRED
H. CHAMBERS AND GeorcE G. Luccuina.* Dept.

AMERICAN PHYSIOLOGICAL SOCIETY 33

of Physiology and Biophysics, Univ. of Vermont

College of Medicine, Burlington.

Potentials were recorded by an amplifier and
oscilloscope from a gold foil electrode on the
round window of an anesthetized cat. The ear was
stimulated by diffuse sound from a loudspeaker
driven by an audio oscillator and amplifier;
gain control settings were constant at all test
frequencies. The tip of a copper wire (14 gauge,
about 2.5 cm long) which was soldered to the apex
of a cone-shaped brass cup was pressed against
the cochlea near the apex of the angle formed by
the bony partition and the edge of the bulla,
anteromedial to the round window. The cup was
filled with alcohol cooled by dry ice to colder than
—40°C. Reduction of potentials was observed
within 2 min. In one instance potentials evoked
by tones of 500 and 1000 cycles were reduced
to 50% of control values within 6 min.; in con-
trast, those evoked by 3000 and 4000 cycles were
reduced by less than 10% of control. Potentials
returned to control values (+10%) within 20 min.
following removal of the cold alcohol. Qualita-
tively similar results were observed in other
instances. (Part of this work was done during the
tenure by one of us of a Lederle Medical Student
Research Fellowship.)

104. Revival of newborn mammals after being
kept in ice for a few hours. M. C. CHane.
Worcester Fndn. for Exptl. Biology, Shrewsbury,
Mass.

Following previous study of the resumption of
heartbeat in the rabbit embryo and fetus (Federa-
tion Proc. 13: 25, 1954; 14: 26, 1955), newborn rats,
rabbits and ferrets were put in a jar and kept at
4°C for $ hr. before being stored in ice. Each
animal was taken out at various intervals, ex-
posed to an infrared light and then artificial
respiration was performed. If revival failed in 1
hr., the heart was exposed, isolated and suspended
in saline to determine the resumption of beat.
All 5 newborn rats revived after the treatment
for 1 hr. After 2-3 hr., 1 of 16 gasped for a few
times and, except for 1, the heartbeat was resumed
in the others. In the newborn rabbit, all 5 revived
after 2-3 hr. After 4 hr., 6 revived and the heart of
10 of 25 animals resumed beating. After 5-6 hr.,
of 16 animals, 1 revived for a few minutes and only
6 isolated hearts resumed beating. After 12-24
hr., only the isolated heart resumed its beat in 9
of 24 animals. In the newborn ferret, all 3 revived
after 1-5 hr. After 12 hr., 2 of 3 revived and the
isolated heart of the other resumed its beat. After
24 hr., only the resumption of heartbeat was ob-
served in 2 treated animals. Respiration and move-
ment were observed at 4°C but heartbeat stopped
after about 1 hr. in ice. The probability of revival
and that of resumption of heartbeat varied con-

siderably between individuals and species. (Sup-
ported by Josiah Macy Fndn.)

105. Evidence for simultaneous lowering of
upper and lower limits of CQ, tolerance.
Joun L. Cuarin (introduced by CLARENCE A.
MaaskeE). Univ. of Colorado Med. Ctr., Denver.
Physiologists have speculated whether ac-

climatization to high CO, extends the upper limit

of CO, tolerance or whether the extent of the CO.
range remains the same but both upper and lower
limits are raised. Experiments of Barbour and

Seevers and of Otis suggest that the range moves

up. There has also been speculation over whether

acclimatization to low COz, which is accomplished
by chronic exposure to altitude, merely reduces
the upper limit of CO, tolerance or whether the
whole range is moved down. Nine subjects resided
continuously at high altitude (Mt. Evans, Colo.,

14,150 ft. and Echo Lake, 10,600 ft.) for 3 wk.

during which their upper limits of CO, tolerance

were measured by ventilatory response to grad-
ually increasing CO: in a rebreathing system and

their low CO: tolerances were measured by a

performance test and by the appearance of tetany,

both during artificially induced hyperventilation.

At the end of the 3 wk. acclimatization period the

subjects returned to Denver, Colo., elevation

5,300 ft., for recovery measurements and the

establishment of normal values. Exposure to the

altitude of Mt. Evans and Echo Lake resulted, in
addition to the well known sensitivity to high CO,
in performance improvement during hyperventila-
tion and almost complete absence of the symptoms
of tetany at those CO: levels which had produced
tingling and twitching during the control period.

These results are interpreted as indicating that

with low CO, acclimatization the CO, range moves

down rather than merely shortening.

106. Steroid-vasopressin interactions. J. J.
CuHart® anpD Rosert Gaunt. Research Dept.,
Ciba Pharmaceutical Products, Inc., Summit,
N.J.

The effect of a series of adrenal steroids, in
varying dosage, was studied on the response to a
standard dose of Pitressin (2 mu/100 gm sub-
cutaneously). The test animals were mature male
rats loaded with 5 ml/100 gm of 0.2% NaCl by
stomach tube. The excretion of water, sodium and
potassium was measured over a period of 180 min.
Hydrocortisone at a dose of 200 ug or higher
antagonized the antidiuretic effect of -Pitressin
and in doses of 1-5 mg actually caused dehydra-
tion; 40 ug were without apparent effect. On the
other hand, doses of 1-5 mg enhanced the natriu-
retic and kaliuretic effects of Pitressin; low doses
were without effect. Prednisolone (Meticortelone)
and prednisone (Meticorten) acted qualitatively

FEDERATION PROCEEDINGS

Volume 15

like hydrocortisone, and any quantitative differ.
ences were minor. Desoxycorticosterone in doses
of 40-200 wg did not affect water excretion ip
Pitressin-treated animals. Doses of 1-5 mg an.
tagonized Pitressin antidiuresis but to a smaller
degree than did the glucocorticoids. Low doges
(40-200 ug) antagonized the natriuretic effect of
Pitressin but doses of 1-5 mg, if anything, aug.
mented it. Kaliuresis was increased only by the
high doses of DC. Aldosterone (Aldocorten) in
doses of 0.064—40 ug produced no clearly significant
modification of the antidiuretic action of Pitressin,
It antagonized Pitressin-induced natriuresis in
amounts of 1.6 wg or higher but had no effect on
potassium excretion in the dosages used. The ratio
of the minimal effective doses of DC and aldos.
terone which would antagonize Pitressin natri-
uresis approximated 25:1. In general, these results
show that corticoids may either enhance or de-
press the actions of vasopressin, the specific result
depending on the nature or dose of the steroid used
and on the variable measured.

107. Mitoses in pinna and _ interscapular
epidermis of mice in relation to physiologic
24-hr. periodicity. A. P. CHaupury,* F,
HALBERG AND J. J. Birrner.* Div. of Cancer
Biology, Dept. of Physiology, Univ. of Minnesota
and Cambridge State School and Hosp., Cambridge,
Minn.

Seven groups, each composed of 18 intact ZBC
mice, were kept under the standardized circun-
stances described elsewhere for studies on daily
rhythm (Journal Lancet 73: 20, 1953). These groups
were killed at 4-hr. intervals, from noon of one
day to noon of the following day. For each mouse,
histologic sections were prepared from the skin
of the interscapular region and pinna and for each
tissue and mouse, 3000 nuclei were examined. In
both tissues, mitotic activity by night was less
than } of the corresponding day value (P < .001).
The mitotic rhythms in pinna and interscapular
epidermis were roughly synchronized with each
other and with the daily rhythm in number of
circulating tail blood eosinophils.

108. Total body electrolyte in the rat. Donal
B. CHEEK* AND CLARK D. West. Dept. of
Pediatrics, Univ. of Cincinnati, Cincinnati,
Ohio.

Total body electrolyte and body weight are
related by an exponential function. These param-
eters, when plotted on an arithmetic grid, fre-
quently show a wide scatter. It is the purpose of
the present study to show that in the rat, total
electrolyte (Na, K, Mg, Ca, Cl and P), water and
nitrogen are related to lean body mass, or to fat
free dry solid by simple linear functions. The
regression equations so derived demonstrate small

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March 1966 AMERICAN PHYSIOLOGICAL SOCIETY 35

standard errors of the estimate and are of use in
demonstrating abnormalities in electrolyte me-
tabolism. From data for total body sodium and
chloride and for intracellular and bone sodium,
identity between the chloride space and extra-
cellular sodium space can be demonstrated. From
the Na/Ca ratio in bone and total body Ca, the
amount of sodium in bone may be calculated.
Similarly from available data, the amount of
intracellular sodium in muscle, liver and red cells
can be approximated. By subtracting the sum of
these values for cell and bone sodium from the
total body sodium and dividing by extracellular
gdium concentration, a volume of distribution is
obtained which is nearly identical to the chloride
space corrected for the chloride in red cells. It
vould appear that the red cells account for most
ifnot all of the intracellular chloride of the body
md that by these techniques body sodium can
be accurately partitioned.

19. Acute hypokalemia and_ ventricular
fibrillation. ABRAHAM CHERBAKOFF AND
Se1icH1t Toyama (introduced by J. W. Rem-
INGTON). Depts. of Physiology and Pharmacology,
Med. College of Georgia, Augusta.

We are testing the postulate that the appearance
of arrhythmias in an ischemic heart is related to
the level of coronary blood potassium. An in-
dwelling catheter was placed in the coronary
sinus and continuous samples were withdrawn for
plasma K determinations. The anterior descending
ramus of the left coronary artery was ligated near
its origin. The K level rose continuously after
ligation, from an initial average of 3.5 mEq/l.
The ECG showed extrasystoles within 2 min.,
md the ventricle fibrillated at about 3 min.,
vhen the K level was 4.3. In another series of dogs,
m infusion of glucose and insulin was given prior
io the ligation. The K level fell from 3.5 to 1.8
mEq/l, the ECG showing the typical changes
wsociated with hypokalemia. Upon coronary
ligation, no arrhythmias were seen until 8 min.,
vhen the K level had risen to 2.8 mEq. By 50 min.,
vhen the K had risen to 4.1 mEq, fibrillation
developed. The differences in the time of arrhyth-
nias, and in the preligation K levels, between the
)groups were significant. At the time of fibrilla-
tion, the K levels in the 2 groups were not different.
tis hoped that the procedure can be altered so
that a low K level can be maintained for a longer
lime after ligation and fibrillation further post-
jned or perhaps prevented. (Cardiovascular
Research Training Program supported by grants
ftom the PHS and the American Heart Assoc.)

ll0. Respiratory adaptations to chronic, high
altitude hypoxia. Huao Cutop1 (introduced
by R. D. Dripps). Inst. de Biologia de la Altura,
Univ. Nacional de Tucuman, Argentina.

Respiratory characteristics, arterial acid-base
equilibrium and arterial oxyhemoglobin dissocia-
tion curves were studied in subjects residing
continuously for many years (residents) at 3990
and at 4515 meters above sea level, and in a group
of lowlanders (newcomers) within 8 wk. after
their arrival at these same altitudes. At 3990 m
average resting pulmonary ventilation (1/min/m?)
was 5.3 + 0.25 in 16 newcomers and 4.5 + 0.14
in 11 residents. At 4515 m 2 newcomers averaged
5.6, and 20 residents 4.9 + 0.10 1/min/m?. In
8 subjects residing and studied at sea level,
ventilation was 3.8 + 0.13 1/min/m?. Alveolar
pCO, alveolar ventilation and oxygen ventilation
equivalent changes were in accordance with total
ventilatory alterations. Oxygen breathing at
both levels of increased altitude depressed average
pulmonary ventilation slightly more in the new-
comers than in the residents, and in no case even
to values associated with air breathing at sea level.
Respiratory response to inhaled CO: was greater
in newcomers and less in residents at high altitude,
than was found in normal subjects at sea-level.
Hemoglobin oxygen affinity and arterial pHs of
residents at high altitude were found to be within
the normal ranges of sea level.

111. Convulsive effects of tripellenamine and
diphenhydramine in monkeys. JosEPH G.
Cuusip, LENORE M. Kopre.orr* anp NICHOLAS
Kopetorr.* Dept. of Bacteriology, Psychiatric
Inst., and Neurological Divs., St. Vincent’s Hosp..,
New York City.

The antihistaminic drugs tripellenamine and
diphenhydramine produced clinical convulsive
seizures in all of 8 chronically epileptic and
5 normal monkeys when administered intra-
venously or intramuscularly in adequate dosage.
Seizures were generally much earlier in onset
following intravenous than following intra-
muscular injection. Tripellenamine usually pro-
voked seizures with smaller doses than diphen-
hydramine similarly administered. The threshold
convulsant dose for epileptic monkeys was gen-
erally significantly lower than that for normal
monkeys. Threshold convulsant values determined
for a) intravenous tripellenamine: epileptic 1-6
mg/kg; normal 5-10 mg/kg 6) intramuscular
tripellenamine: epileptic 0.5-6 mg/kg; normal 16
mg/kg c) intravenous diphenhydramine: epileptic
1-12 mg/kg; normal 20 mg/kg d) intramuscular
diphenhydramine: epileptic 4-24 mg/kg; normal
30 mg/kg. Activation of a spike focus in the EEG
of an epileptic monkey sometimes occurred follow-
ing parenteral antihistaminic treatment; in other
monkeys a significant change in EEG could not be
detected until the actual onset of clinical seizures.
Fatal reactions occurred in 4 of the 13 monkeys
tested which was qualitatively similar to those

36 FEDERATION PROCEEDINGS

reported following toxic doses in other species
(mice, rats, rabbits, guinea pigs, dogs and mice).
The potential hazards of the use of large parenteral
doses of these antihistaminic drugs is emphasized.
(Aided by a grant from the Natl. Insts. of Health,
PHS.)

112. Chemical analyses of human _ post-
mortem tissues as aid in determining
physiological status of flying personnel
prior to aircraft accidents. RoBeRtT T. CLARK,
Jr.,8.S. Witks* anp Donatp D. Van Fossan.*
Dept. of Physiology-Biophysics, USAF School of
Aviation Medicine, Randolph Air Force Base,
Texas.

Methods of estimating the possibilities of the
presence of hypoxia prior to a fatal accident have
been applied to Air Force pilots in fatal crashes.
The method consists of the measurements of lactic
acid in brain and spinal cord as a test for hypoxia
due to oxygen-lack and carbon monoxide con-
centrations in all tissues that are available. Sixty-
six cases from aircraft accidents have been studied.
Brain tissue from 20 of these cases has been
analyzed for lactic acid. Ten showed lactic acid
values to indicate hypoxia (above 180 mg%) prior
to death. Results from tissue analysis of the 66
cases indicated 27 with blood carbon monoxide
values above 30% COHb. Control specimens have
been obtained from local hospitals.

113. Central localization of the osmotic con-
trol center. NEvILLE P. CLarKE, GEorGE D.
ZUIDEMA AND Mary F. Minton (introduced by
Raymonp F. Kune). Aero Med. Lab., Wright
Air Develop. Center, Wright-Patterson Air Force
Base, Ohio.

Experimental studies in the osmotic control of
body fluid volume were carried out by intra
carotid injection of hypertonic solutions in con-
scious, highly-trained animals. The resulting
antidiunetic responses were interpreted as evi-
dence for a central ‘osmoreceptor’ center, (VER-
NEY, 1947). Similar experiments in our laboratories
in animals under light chloralose anesthesia have
confirmed the oliguria which follows such injec-
tions. These studies have been expanded to include
a series of injections into a cannulated femoral
artery to determine if the pain accompanying such
an injection could act as a nonspecific stimulus
and result in oliguria, either by a neural or hor-
monal mechanism. Femoral artery injections
equal in concentration and duration to those used
for intracarotid injection failed to produce an
antidiuresis. On the contrary, in all cases except
the normal saline controls, a diuresis ranging
from 14 to 60% resulted. Defensive motions of the
hind limb and hyperventilation denoted intense
local irritation from the injections of sodium

Volume 4 Ma

sulfate solution. These diuretic responses resulting wa}
from femoral artery injection were shown to bef con
due to osmotic diuretic action of the solute acting § var
on the renal tubule. In the case of carotid arter§ pro
injections, release of antidiuretic substance was volt
sufficient to mask this diuretic effect. sim

114. Relations between EEG changes and
experimental local closed cerebral injury
with edema. RaymMonp CLASEN, PAULINE the
CookE, JAMES WILLIAMS AND GEORGE Hag},
(introduced by C. Bruce Taytor). Rush Lab, of
Pathology, Presbyterian Hosp., Chicago, Ill. and
Closed unilateral circumscribed lesions wer

produced over the convexity of the cerebrum in} f ij,

nembutalized dogs by a cold instrument applied
to the external surface of the intact calvarium, +43

EEG recordings of the experimental and con-] x

tralateral control hemispheres were made befor,

during and at intervals up to several hours after
production of lesions. Cisternal pressure was} soy
continuously recorded. After each experiment the} iq
water content of the damaged and control hemi-} me
spheres was determined separately. The blood rep
content of each hemisphere was then calculated] pag
from comparative analyses of the iron contents of
venous blood and dry residues of each hemisphere,

EEG tracings disclosed that the immediate locus} 116.

of cerebral damage promptly became electrically} al

inactive and remained unchanged thereafter} Ji

Electrical activity at a distance from the im] F

mediate site of injury underwent a series of vari-| P

able though less profound changes which were doe:

not always related either to the early rise in} blo
cisternal pressure or to subsequent fluctuations inf acic
pressure. However, though there was some retum| dete
of electrical activity toward normal after the} mte
initial insult, an abnormal plateau state slowly{ gasi
developed. Following this, there was a secondaty] wer
decline which appeared to parallel the develop-§ anti
ment of an increased water content rather than} hyd
blood content of the damaged hemisphere. EEG
changes in the damaged hemisphere were at time

accompanied by changes in electrical activity o

the contralateral undamaged hemisphere.

115. Relationship between respiratory dead:
space and resistance and estimation of lung
tissue resistance. JoHN A. CLEMENTS AND
Rupo.tpw P. JoHNson (introduced by W. H.
CHAMBERS). Chem. Corps Med. Labs., Army
Chem. Citr., Md.

In a previous communication we reported that
intense bronchoconstriction induced by an anti
cholinesterase agent, DFP, in dogs maintained
constant tidal-volume artificial ventilation wa
accompanied by major changes in respiratory
deadspace and resistance. These changes sug¢fforc
gested that under these circumstances lung-ait{ the

7olume f§

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PAULINE
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alvarium,
and con-
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urs after
sure was
ment the
rol hemi-
he blood
alculated
ntents of
nisphere,
ate locus
>ctrically
.ereafter,
the im-
3 of vari-
ich were
y rise in
ations in
ne returm
ifter the
e slowly
econdaty
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at times
tivity of

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NTS AND
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March 1956

way resistance could be represented by a constant
component (tissue viscous resistance) and a
yariable component (airway resistance, inversely
proportional to the square of the deadspace
yolume). Repetition of these experiments with
smultaneous estimation of functional residual
yolume and of evenness of lung ventilation has
suggested that these changes are related to de-
geases in airway caliber distributed throughout
the tracheobronchial tree, rather than to reduc-
tion in the actively ventilated fraction of the
lungs. Simultaneous measurements of resistance
and deadspace in normal men showed these
quantities to follow the same mathematical
relationship when lung volume was varied through
awide range. Resistance at small lung volume was
444 times that at vital capacity and deadspace
at small lung volume was 40-50% of that at full
lung volume. On the assumption that lung tissue
resistance was substantially constant, these data
could be extrapolated to zero airway resistance
and estimates of lung tissue resistance obtained.
These were in good agreement with the values
reported by McIlroy, Mead, Selverstone and
Radford, but exceeded values calculated from the
method of repetitive interruption of flow.

ll6. Barrier offered by gastric mucosa to
absorption of sodium. CHARLES F. Cops,

Joun A. Hiaeins* anp ALAN L. Orvis.* Mayo

Fndn. and Mayo Clinic, Rochester, Minn.

Prior studies have demonstrated that sodium
does not pass from the gastric contents into the
blood stream if the gastric mucosa is secreting
aid. This observation has been extended by
determining, under a variety of circumstances, the
tate of passage of labeled water and sodium from
gastric contents to blood in dogs. The animals
were under pentobarbital anesthesia with the
antral mucosa excluded by ligation. Addition of
hydrochloric acid to neutral gastric contents
stopped the absorption of sodium in precisely the
same manner as did stimulation of the secretion of
wid. If, during the continuous production of acid
by stimulation with histamine, when no sodium
was passing out of the stomach, the contents were
neutralized by sodium hydroxide, then sodium
left the gastric contents and passed into the blood
stream. To our surprise, however, reacidification
id not then block the absorption of sodium.
The latter result suggested, then, that the deter-
mining factor in the passage of sodium from gas-
tric contents to blood stream might be the relative
toncentrations of sodium and hydrogen ions
and not the pu. Critical tests have shown that this
is the case. In the presence of either intrinsic or
extrinsic hydrochloric acid, radiosodium may be
forced from the contents into the blood stream by
the addition of sodium chloride, although the

AMERICAN PHYSIOLOGICAL SOCIETY

concentrations necessary to produce the move-
ment are very high, Throughout the tests the rate
of passage of water from gastric contents to blood
stream was practically unaffected by any of the
changes in the contents. Thus, under the circum-
stances of these tests the absorption of sodium
and water proceeded independently.

117. Avoidance learning motivated by hypo-
thalamic stimulation. Bertram D. CoHEn,*
GrorcGE W. Brown AND MagJori£ L. Brown.*
General Med. Research Lab., VA Hosp., and
Dept. of Surgery, Univ. of Iowa Med. School,
Iowa City.

Two experiments were performed to investigate
the conditionability of emotional behavior
aroused by electrical stimulation of the hypo-
thalamus in cats. All animals were prepared with
chronically implanted electrodes. In the first
experiment (classical conditioning), hypothalamic
shock was delivered during the last 2 sec. of a 10-
sec. 5000 cps tone. The cats acquired a strong
tendency to orient to the locus of the tone source.
This was interpreted as a conditioned preparatory
response. Appropriate control groups were run.
In the second experiment (instrumental learning),
a series of cats were trained to avoid hypothalamic
stimulation by jumping a hurdle during a 15-sec.
warbling tone. If the cats failed to cross the
barrier, the hypothalamic stimulus was delivered
and was continued until they did cross. If they
crossed during the 15-sec. tone period, hypo-
thalamic shock was omitted. Twenty trials per
day were run for 10 days with 2-min. intertrial
intervals. During the early stages of training the
initially ‘disorganized’ hypothalamic behavior
acquired a more efficiently organized functional
form (escape). Following this the animals learned
to respond adaptively to the tone (avoidance).
Thus, hypothalamic stimulation was effective in
providing a motive for learning similar to that
ordinarily provided by peripheral pain stimula-
tion. (Supported in part by a research grant
M-641 from the Natl. Inst. of Mental Health.)

118. Interrelations among acetoacetate, in-
organic phosphate and inorganic sulfate
during their renal tubular reabsorption in
the dog. Jutius J. CoHEN AND FREDRIK BERG-
LUND. (introduced by Rospert A. Kenor). Dept.
of Physiology, Univ. of Cincinnati College of
Medicine, Cincinnati, Ohio.

In a recent communication from this laboratory
it was shown that glucose depresses, while
phlorizin enhances the renal tubular reabsorptive
maxima (Tm) for acetoacetate, phosphate and
sulfate. These effects of glucose and phlorizin are
more easily elicited for phosphate and sulfate than
for acetoacetate. The relative magnitude of the

molar Tm for each of the 3 ions was postulated to
be the reason for the differences in response to
glucose and phlorizin. Experiments were designed
to determine: 1) whether the Tm of the more
rapidly reabsorbed substance (acetoacetate) is
less subject to change due to its greater affinity
for the reabsorptive pathway which the 3 ions
share and 2) whether the 2 less rapidly reabsorbed
substances (sulfate > phosphate) show any com-
petitive interrelationships. These experiments
revealed the following: Tm-acetoacetate is un-
affected by raising the filtered loads of phosphate
or sulfate; both Tm-phosphate and Tm-sulfate are
depressed by high filtered loads of acetoacetate;
Tm-phosphate is unaffected by increasing the load
of sulfate; in contrast, Tm-sulfate is depressed by
a rise in the amount of phosphate filtered. Since
Tm-sulfate is higher than Tm-phosphate, these
findings indicate that the relative magnitude of
the molar Tm is not a definite indication of the
affinity of the anion for the reabsorptive pathway.
Further evidence is presented that it is the rela-
tive position of the anions in the lyotropic series
which is one of the rate-limiting factors in tubular
reabsorption.

119. Analysis of position sense in humans.
LEoNARD A. CoHEN (introduced by C. H. W.
RuuweE). School of Medicine, Univ. of Pittsburgh,
Pittsburgh, Pa.

The accuracy with which a temporarily sightless
person can point to a reference spot on a target
was investigated in human male and female
subjects. Since the experimental arrangement
required that the arm and hand of the subject be
fully extended, the test was principally a measure
of position sense in the shoulder. As a control,
subjects were asked to point to a reference spot
without being deprived of eyesight in order to
test motor accuracy. Since every subject was able
to place his finger exactly on the reference point
under these conditions any inaccuracy in pointing
which occurred while the subject’s eyes were
closed was due to the limitations of his shoulder
position sense. It is felt that shoulder joint
proprioception was being measured principally
rather than arm muscle proprioception because
only the former is always a measure of arm posi-
tion and the existence of joint proprioceptors is
now well established. The data were analyzed to
see if any special shoulder angles were signifi-
cantly more accurate in a given subject than were
other angles. The attempts at duplicating a given
standard point were examined to see if the subject
usually made the same type of error, e.g. over-
shooting the point. Some subjects whose accuracy
differed considerably were retested several times
to determine the amount of variation in a given
individual and also to see if 1 subject was con-
sistently more accurate than another.

FEDERATION PROCEEDINGS

Volume ij

120. Effect of 2,4 dinitrophenol on the hepatic
clearance and extraction of Rose Bengal in
dogs. B. Compes, H. O. WHEELER, A. VW,
Cups anp O. L. Wane (introduced by H, B,
vAN Dyke). Dept. of Medicine, Columbia Univ,
New York City.

Rose Bengal (RB, tetraiodo-tetrachlor fluo.
rescein) appears to be almost completely bound
by plasma proteins. Less than 1% could be re.
moved by dialysis from normal human plasma
and purified serum albumin solution (4.2 gm %)
containing the dye in concentrations of 20-2
mg %. It appears to migrate chiefly with the
albumin fraction during paper electrophoresis,
Twenty minutes after administration of a ‘priming
dose’ (10-20 mg) and sustaining infusion (0.35
1.09 mg/min.) of RB to 11 nembutalized (3
mg/kg) dogs, femoral arterial and hepatic venous
blood samples were obtained at intervals. The
mean value for hepatic clearances of RB was
ml/10 kg/min. + 8.D. 25 ml. Hepatic blood flow,
calculated from the hepatic clearance and extrae-
tion of RB averaged 326 ml/10 kg/min. + 8.D,
93 ml. The mean hepatic extraction of the dye was
30.7% and there was no evidence of extraction
elsewhere. In order to determine whether the
hepatic removal of Rose Bengal is dependent on
aerobic phosphorylation, the effect of 5-10 mg/kg
of 2,4 dinitrophenol was studied in 5 dogs. The
mean hepatic removal rate of 0.815 mg/min. was
not significantly different from the control value
of 0.881. RB clearance decreased, however, in all
dogs from a mean value of 64 ml1/10 kg/min. to
47 ml. Mean hepatic blood flow did not change,
but hepatic RB extraction fell from 39.6 to 30.5%
and arterial RB concentration rose from 1.227 to
1.538 mg%. These data suggest that the ability of
the liver in anesthetized dogs to remove RB from
the blood may be slightly impaired by 2,4 dinitro-
phenol.

121. Effects of potassium ions on recovery
processes of nerve. C. M. ConneELLy,* W. J.
SuLLIVAN* AND D. W. Bronx. Rockefeller Inst.,
New York City.

Metabolic and electrical variables associated
with activity and recovery therefrom have been
measured in frog nerve after removal of the
perineurium. Virtually all of these are significantly
changed as the concentration of K+ in the bathing
solution is varied over the range 0-8.5 mm. The
properties measured during activity (0.5-1 hr.
at 50 volleys/sec.) and during recovery were in-
crease in oxygen uptake, amplitude of action
potential, refractory period and development
of positive increment in membrane _ potential
(positive after-potential, observed once/min.
during a 5 sec. interruption of tetanus). Nerves in
potassium-free solution, compared with nerves in
2.1-5 mm K*, show a reduced capacity for sustained

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March 1956

activity and, recoveries in all measured variables
so slowed that they are incomplete even after 3
br. In contrast, nerves tetanized in 8.5 mm Kt
show little change in action potential or refractory
period (or slight increase and decrease, respec-
tively) and sustain a large positive after-potential ;
recovery is rapidly completed in 40-50 min. In
0-8.5 mm K*, the magnitude of the positive after-
potential was 10-20% of the potential change
observed upon depolarization of the nerve by 120
mu K+. Our observations suggest that the posi-
tive after-potential is an electrical sign of ion-
transport processes which gradually restore the
ion balance characteristic of the nerve at rest,
the level of extracellular K* largely determining
the kinetics of that restoration and the oxygen
uptake closely reflecting the energy demand of the
transport processes.

122. Increased growth hormone activity in
plasma of pregnant rats. ALEx N. ContTo-
pouLos* AND Mrrram E. Simpson. Inst. of
Exptl. Biology and Dept. of Anatomy, Univ. of
California, Berkeley.

The presence of growth promoting activity in
the plasma of normal and pregnant rats was in-
vestigated. Rats were bled through the aorta on
days 16-17 and 20-21 of pregnancy. The plasma
was injected in different amounts into recipient
hypophysectomized rats for 4 or 14 days. Plasma
from normal rats was injected in the same volumes
and for the same period into other groups. Un-
injected hypophysectomized rats served as con-
trols. The criteria for the presence of growth
activity in the plasma were: increase in the width
of the uncalcified portion of the proximal epiphys-
eal cartilage of the tibia, tail length, and body
weight increase. A straight line relationship
between dose and response was found by all these
criteria for plasma of both normal and pregnant
tats. The plasma from pregnant rats contained
three times the amount of growth activity present
in normal rat plasma. No difference in the growth
promoting activity was found in the plasma
from the two different periods of pregnancy. This
growth promoting activity was not decreased by
hypophysectomy of the mother at the 12th day
of pregnancy. The role of the fetal pituitary and
the placenta in the production of growth hormone
during pregnancy will be discussed. (Aided by
PHS Grant A-661(C) and A-662(R).)

123. Effect of changes in tidal volume and
alveolar pCO, on physiological dead space.
D. Y. Cooper anp C. J. LAMBERTSEN (intro-
duced by I. S. Ravp1n). Dept. of Pharmacology
and Harrison Dept. of Surgical Research, Univ. of
Pennsylvania School of Medicine, Philadelphia.
Physiological dead space, calculated from

Measurements of arterial pCO2z, was previously

AMERICAN PHYSIOLOGICAL SOCIETY

reported to be markedly increased by CO: in-
halation (Federation Proc. 12: 28, 1953). This
investigation attempts to separate influences of
tidal volume and alveolar pCO: upon dead space.
Dead space changes, calculated from alveolar or
arterial pCO., were determined during several
levels of voluntary hyperventilation, exercise
hyperventilation, and increase in tidal ventilation
without alteration of alveolar pCO2. Increase in
tidal volume alone (alv. pCO. 39 mm Hg) of 1
liter enlarged dead space 106 cc. With the same
tidal volume change dead space increases were 100
ce during exercise (art. pCO: 39 mm Hg), 258 ce
during CO, breathing (art. pCO, 51 mm Hg), and
56 ce during voluntary hyperventilation (alv.
pCO, 30 mm Hg). With alveolar pCO: constant,
dead space increased 11 cc/100 cc increase in
tidal volume. Deducting tidal volume effect from
dead space in CO; breathing and voluntary hyper-
ventilation indicated that, at constant tidal
volume, hypercapnia enlarged dead space about
30 cc/mm Hg while hypocapnea diminished dead
space about 3 ec/mm Hg. It is suggested that a
pharmacodynamic effect of altered pCO: is nor-
mally algebraically additive with a separate,
mechanical effect upon dead space of altered tidal
volume.

124. Renal clearance of radiocarbon-labeled
inulin carboxylic acid. J. A. D. Cooprr,*
B. B. Rees,* N. 8. Raprn* anp D. P. Ear.e.
Depts. of Biochemistry and Medicine, North-
western Univ. Med. School and Radioisotope Unit,
Veterans Admin. Research Hosp., Chicago, Til.
A method has been developed for the estimation

of radiocarbon-labeled inulin carboxylic acid

(ICA) in biological fluids. The ICA in plasma or

urine is decarboxylated quantitatively with silver

persulfate at room temperature. The radioactive
carbon dioxide formed is trapped in a methanol
toluene solution of a high molecular weight
quaternary amine (Hyamine) using a simple

diffusion apparatus constructed from two 50-ml

Erlenmeyer flasks. The radioactivity of the

quaternary amine carbonate solution is measured

with a liquid scintillation counter employing a

toluene diphenyloxazole system. The renal clear-

ance of ICA was compared to simultaneous stand-
ard creatinine and inulin clearances in 3 dogs. Ten
studies, each with 3 consecutive 15-min. clearance
periods, were made. The agreement between the

3 methods was good for the individual clearance

periods. The average values of the 30 periods were

75.80 ml/min. for ICA, 72.52 ml/min. for creatinine

and 75.70 ml/min. for standard inulin. The clear-

ances obtained by the 3 techniques were compared
by regression analysis. The slopes of the 3 possible
regressions were not found to differ significantly
from unity. The standard error of estimate for
creatinine vs. inulin was +5.38; for inulin vs.

40 FEDERATION
ICA, +7.10 and fer creatinine vs. ICA, +7.47.
(Aided by grants from the Public Health Service
and the Chicago Heart Assoc.)

125. Vascular reactivity to epinephrine fol-
lowing sympathectomy. T. Cooper, V. L.
WiLiMAN AND A. B. Hertzman (introduced by
A. W. Ricuarpson). St. Louis Univ., St. Louis,
Mo.

The reactivity of the superficial vessels of the
hind footpad of the dog to epinephrine after
lumbar ‘sympathectomy’ (histologically verified
removal of the lumbar sympathetic chain from
above the renal vessels to below the sacral prom-
inence) was evaluated by photoelectric plethysmog-
raphy. Empirical correlations between the
amplitude of the volume pulse and blood flow, and
theoretical considerations indicate that at a con-
stant pressure pulse, the 4th root of the amplitude
of the volume pulse is related to the length of the
circular smooth muscle in the walls of the minute
blood vessels. Analysis based on this relationship
demonstrated no significant difference in the re-
activity of the vessels on ‘sympathectomized’ and
the intact sides to L-epinephrine bi-tartrate in
doses of .01 y/kg body weight given intrave-
nously. This dose range, slightly above threshold,
was selected because it consistently elicited con-
striction in this vascular bed without inducing
gross changes in central hemodynamics. However,
the pad vessels of limbs which had been totally
denervated 8-14 days before testing showed an
augmented reactivity to L-epinephrine in doses of
01 y/kg. Tetraethylammonium chloride (TEA) in
doses of 7 mg/kg elicited less dilatation on the
‘sympathectomized’ side than on the intact side.
Also failure of the pad vessels to dilate in response
to TEA occurred more often on the ‘sympathec-
tomized’ side. Results will be discussed in relation
to structure, innervation and threshold of the
pad vessels to epinephrine. (Aided by St. Louis
Heart Agsoc. and PHS.)

126. Yields of isolated rabbit, hamster and
human platelets obtained with new micro-
method. ALFRED L. CopLtey, TRAJAN BaLEA*
AND Vo-VinH-Hoa.* Research Labs., Centre
National de Transfusion Sanguine and Institut
National d’Hygiéne, Paris, France.

The application of the Copley-Houlihan macro-
method for platelet isolation to 5 cc blood resulted
in high yields but was not suitable for 0.1 cc
samples. A micromethod was therefore developed.
Anticlotting fluid (A) was composed of 9 volumes
0.5% Versene (disodium ethylene diamine tetra
acetate) in 0.85% NaCl, px 7, to which, before
usage, 1 volume 2% Triton WR 1339 (an alkyl aryl

PROCEEDINGS

Volume ij

polyether alcohol) was added. Washing fluid (8)
was 0.9% NaCl solution, pH 7. With special 2 ¢&
diluting pipette, blood from rabbits, hamsters and
men was drawn to 0.1 ce mark, followed by fluid 4
to the 2 ce mark. Contents of pipette were centri.
fuged 15 min. at 1500 rpm and 4°C. The super.
natant was decanted and platelet sediment washed
with fluid B, centrifuged at 1500 rpm at 4°C. After
2 more washings sediment was suspended in 2 ¢¢
anticlotting fluid (A), followed by platelet count,
Yields of isolated platelets, based upon initial and
final Brecher-Cronkite platelet counts, ranged
from 39 to 71 and averaged 47% in 30 subjects,
Final platelet suspensions, silky smooth in ap.
pearance, ranged from 6000 to 37,000 per mm,
High platelet counts, up to 1,460,000, were fre.
quently found in peripheral rabbit blood. The
highest yield compared to original count of
420,000 and lowest yield to 1,200,000. Yields from
0.1 cc blood samples must be considered high when
compared to macromethods in which 10-400 ec
blood are employed. The micromethod makes it
possible to secure cutaneous blood samples from
small laboratory animals, infant and adult human
subjects with not readily accessible veins for re-
peated isolations to be used in various studies
(CopLEY AND Bafa. Sang 26: 841, 1955) on
platelets. (Aided by a grant from the Natl. Science
Fndn., Washington, D. C.)

127. Comparative studies on different bae-
terial luciferase preparations. Mitton J,
CorMIER (introduced by C. W. SHEPPARD),
Oak Ridge Nail. Lab., Biology Div., Oak Ridge,
Tenn.

Various bacterial luciferase preparations were
compared for their ability to oxidize DPN
in the presence of quinones and ferricyanide and
for their ability to oxidize DPNH: for light pro-
duction. From such studies, it was concluded that
luciferase is not a DPNH: oxidase and that the
only apparent function of the enzyme is that of
light production. Various crude preparations of
luciferase from A. fischeri and from an unidentified
coccobacillus (Gest) were obtained by extracting
the acetone-dried powder or by lysing the cells.
Purification of the different types of crude luci-
ferase preparations by (NHj,).SO, fractionation
and starch-column electrophoresis resulted in an
enzyme which would give light in the presence of
DPNH2, FMN, long-chain aldehyde, and Oz only
when DPNH:z oxidase obtained from E. coli was
added. Some luciferase preparations from A.
fischeri showed an absolute requirement for al-
bumin before light was emitted in the presence of
FMNH; and aldehyde, whereas others gave light
in the absence of albumin. The nature of activa-

olume If

luid (B)
cial 2 ¢
ters and
y fluid A
e centri-
€ super-
| washed
C. After
1 in 2¢¢
t count,
itial and

ranged
subjects,
1 in ap-
er mm’,
vere fre-
od. The
ount of
lds from
gh when
+400 ee
nakes it
les from
t human
s for re-

studies
955) on
Science

it bae-
LTON J,
/PPARD),

k Ridge,

ns were
DPN;
ide and
zht pro-
led that
chat. the
that of
tions of
lentified
tracting
re cells,
de luci-
onation
d in an
sence of
Oz only
coli was
rom A,
for al-
sence of
ve light
activa-

March 1956

tion by albumin apparently lies, for the most part,
in the removal of heavy metals. The luminescence
of purified luciferase preparations in the presence
of FMNH:2 was strongly inhibited by p-chloro-
mercuribenzoate, indicating that luciferase is
probably a sulfhydryl enzyme. Ferric and ferrous
jron inhibited luminescence but not nearly so well
as the corresponding 8-hydroxyquinoline chelates.
Hemin was found to combine with luciferase and
jg one of the most potent inhibitors of cell-free
bacterial luminescence known.

128. Association of anemia and hyperlipemia
with experimental hypoproteinemia. Sam-
veL A. Corson and ExvizaBetu O’LEARY Cor-
son.* Depts. of Physiology and Pharmacology
and of Psychiatry, Univ. of Arkansas School of
Medicine, Little Rock.

Hypoproteinemia was produced in trained un-
anesthetized mongrel dogs by a combination of
massive plasmapheresis and a low protein diet
designed to provide a minimum quantity of
essential amino acids, a reasonably low ratio of fat
to carbohydrate, sufficient roughage and palat-
ability. This diet (35 gm/kg/day) provided daily
70 C and 0.3 gm of protein (of high biological
value)/kg of body weight. This method per-
mitted the production of a fairly constant degree
of hypoproteinemia without the necessity of
resorting to frequent plasmapheresis. The plasma
proteins were reduced from an average normal
value of 6.6 gm% to an average figure of 3.4
gm%. The hypoproteinemic animals showed
significant increases in extracellular space (thio-
cyanate method), evidenced as pitting edema in
most cases. In all dogs the hypoproteinemia was
invariably accompanied by a persistent anemia
and a sporadic or persistent hyperlipemia. The
hematocrit reduction was more intense than that
of the plasma proteins. Thus, while the maximum
plaama protein reduction amounted to about
0%, the hematocrit was reduced to about 30%
of the normal value. The reduction in hematocrit
would continue even while the plasma protein
concentration would rise. This would occur even
when more erythrocytes were infused than were
removed by plasmapheresis. These hematocrit
changes were not due to alterations in plasma
volume (T-1824 method). The hyperlipemia was
remarkable for the fact that in some instances it
would suddenly appear and disappear within
periods as short as 20 min. (in dogs fasted for at
least 18 hr.). In other cases the hyperlipemia
would persist for several days. (Aided in part by
grants from the Natl. Insts. of Health, PHS.)

29. Spatially oriented heterogeneity of
chloride exchange across epithelial cell

eavrrry i

AMERICAN PHYSIOLOGICAL SOCIETY 41

surfaces of gastric mucosa. ERNEST Cor-

LOVE* AND C, ApRIAN M. Hoasen. Natl. Heart

Inst., Bethesda, Md.

Cells of glands of external secretion have the
unique capacity to cause net, unidirectional
transfer of substances across their opposite
nutrient and secretory surfaces. The present study
attempts to define the 4 individual flux rates of
radioactive chloride into and out of the opposite
surfaces of the epithelial cell layer of the isolated
frog gastric mucosa, which has been shown to
produce active, trans-mucosal transport of
chloride from nutrient to secretory side (HoGBEN,
Am. J. Physiol. 180: 641, 1955). Introduction of
Cl-36 into nutrient bathing solution of one-half
of the mucosa and into secretory solution of other
half permitted steady-state analysis from
measurement of the 2 trans-mucosal fluxes, the
specific activity of solutions of origin and of
mucosal cells. C-14 carboxyl-labeled inulin was
used to measure mucosal extracellular spaces
accessible from nutrient and secretory surfaces
(averaged 54% and 3% of wet weight, respec-
tively). Results indicate that the exchange rate of
chloride at the secretory surface of the epithelial
cell layer is much greater than at the nutrient
surface, since the specific activity of cell chloride
relative to solution of origin was high when Cl-36
was introduced into secretory solution and low
when introduced into nutrient solution. This
finding has a bearing on the mechanism of chloride
transport and affects interpretation of measure-
ments under some conditions of exchange rate and
extent of exchangeability of secreting tissues.
Data also showed: a) complete exchangeability of
gastric intracellular chloride; b) kinetics of inulin
distribution and c) intracellular cation compo-
sition (predominantly K, negligible Na).

130. Chemical regulation of heat production
in cold-adapted rats. WALTER CoTTLE* AND
Loren D. Cartuson. Dept. of Physiology and
Biophysics, Univ. of Washington School of
Medicine, Seattle.

The capacity of cold-adapted rats (5°C) to
increase heat production by chemical-regulatory
mechanisms was tested in animals whose muscular
activity was blocked by curare. Following tracheal
cannulation for artificial ventilation and injec-
tion of curare, animals were kept at a room tem-
perature of 30°C for 1 and 3 hr., after which the
temperature was gradually lowered to 5°C over
a period of 4 hr. and maintained at 5°C for 2 hr.
Oxygen consumption, rectal, foot and air tem-
peratures, heart rate and muscle activity were re-
corded throughout.

Warm-Adapted Cold-Adapted
Intact rats Smallincreasein 100% increase in
Oz uptake. Oz uptake.
Marked drop Little or no
in rectal temp. drop in rectal
temp.
Adrenal-de- Similar to intact Less increase in
medul- rats O2 uptake and
lated rats greater drop

in rectal temp.
than in intact
No increase in
Oz uptake.
Marked drop
in rectal temp.

No increase in
O2 uptake.
Marked drop
in rectal temp.

Spinal rats
(no cur-
are)

Chemical regulation of heat production is greatly
enhanced by cold-adaptation. Reduction of the
calorigenic response of adapted animals by adrenal
medullectomy suggests that this response is
mediated by epinephrine released by the medulla
as well as by sympathetic endings. More of the
calorigenic hormone may be released by the cold-
adapted rats and/or such rats may be more
sensitive to the hormone.

131. Some relationships of caudate nucleus to
cerebellum. WiLi1am 8S. Coxre* anp Ray §.
Sniper. Depts. of Surgery and Anatomy, North-
western Univ. Med. School, Chicago, Ill.
Measured electrical pulses were applied to head

and body of the caudate nucleus, and cathode ray

tracings of the evoked responses in the cerebellum
were recorded. Stereotaxic placement of elec-
trodes was carefully controlled by histological
procedures. The following cerebellar areas were
studied: culmen (ant. lobe), lobulus simplex,
paramedian lobule, crus I and II and tuber vermis.

Although all of the above areas responded in

some animals, the most persistent responses were

observed in crus I and II and tuber vermis. Data
collected on crus I are confirmatory of Walberg’s

1954 aifatomical studies. Since positive observa-

tions can be made following transection of the

pons, the role of the inferior olive as an inter-
mediate nucleus is being considered (also in agree-
ment with Walberg’s studies). It is believed that
this represents a means for interaction between
the basal ganglia and cerebellum. The usual wave

form shows 2 peaks—one at approximately 5

msec. and the other at 12-15 msec. All studies

were made on cats under ether and d-tubo-cu-
rarine anesthesia. Electrical pulses were applied

via a Grass S4A stimulator, amplified through a

Tektronix preamplifier and visualized on a Du-

Mont 322 cathode ray unit. (Research aided by a

grant from PHS.)

132. Influence of dietary carbohydrate on the
development of fasting diabetes in man.

FEDERATION PROCEEDINGS

J. W. Craig, M. Miuter, W. R. Drucker ay)
H. Woopwarp, JR. (introduced by R. A. Sup.

LEY). Dept. of Medicine, Western Reserve Unin§:

Cleveland, Ohio.

Hill, Baker and Chaikoff (J. Biol. Chem, %y
705, 1954) concluded that the type as well as t
quantity of dietary carbohydrate may influeng
the rate of utilization of subsequently admin.
istered glucose. Fructose feeding in rats producej
an impairment in oral glucose tolerance almost y
great as with fasting. This was ascribed tj
impaired hepatic glucokinase activity, a magi.
festation of enzymatic adaptation to diet. }
the phenomenon demonstrated in rats occurred iy
man, it might explain the development of huma
fasting diabetes. The present experiments wer
designed to determine whether or not dietary
fructose altered glucose tolerance in man. Fo
normal subjects were fed a diet whose sole carho-

hydrate was 200 or 250 gm of fructose daily forjf:

days, after which an intravenous glucose tolerance
test was performed. Tolerance tests were per.
formed in the same subjects after 3 days on a die
whose sole carbohydrate was the same quantity
of glucose. In these subjects, fructose feeding
produced no significant alteration in intravenow
glucose tolerance. In similar experiments, »
impairment of oral glucose tolerance was demo-
strated after 1 day of fasting and 3 days of fructose
feeding. Failure to find an altered glucose toler.
ance after fructose feeding suggests that the
lowered concentration of glucose in portal ven
blood, which is present in man as well as the mi
during fructose feeding, is not the determining
factor in the production of starvation diabetes in
man. Further experimentation
clarify the role of liver glucokinase in this phe
nomenon.

133. Pathogenesis of experimental hyper.
tension in dogs produced by carotid sinw
area constriction. E. CRANDALL* AND G., f.
WakERLIN. Univ. of Illinois College of Medicim,
Chicago.

Previously, our research group reported that
bilateral constriction of the carotid sinus ares,
including the internal carotid and external carotil
(and occipital) arteries, in the dog resulted i
hypertensions of 30-100 mm Hg in 9 of 10 dog
(Circ. Research, 2: 416, 1954). Presently, thes
hypertensions have durations of 18-31 months and
have been confirmed in 3 additional dogs. General
health, appetite, body weight, body temperatur,
urinalyses and heart rate have continued norma.
Passive transfer of large doses of homologots
antirenin to hog renin (which produces a striking
antihypertensive effect in renal hypertensive
dogs) was without effect on carotid sinus are
hypertension. Dibenzyline (2 mg/kg i.v.) pr

Volume iif!

is needed tf;

duce
tens

pres
dati

tens

Volume i March 1956
duced an acute reduction in pressure to normal
tension. Chronic treatment with Dibenzyline for
2? months showed only moderate reduction in
pressure. In further experiments aimed at eluci-
dating the pathogenesis of sinus area hyper-
tension, bilateral constriction of the vertebral
arteries in 3 dogs resulted in normotension in 2
and hypertension of 40 mm Hg in 1. Bilateral
constriction of the internal and external carotid
(and occipital) arteries above the sinus in 2 dogs
produced hypertension, as did bilateral constric-
tion of the denervated sinus in 1 dog. Three of 4
dogs subjected to bilateral removal of the carotid
sinus developed modest hypertensions of 30 mm
Hg. These preliminary findings suggest that
pathogenesis involves an alteration in cerebral
hemodynamics with both increased vasoconstric-
tor nerve tonus and a humoral factor. (Aided by

JCKER Ay)
. A. Sap.
2rve Unit,

Them. Mh
vell ag
~ influeng
ly admin.
| producef
almost 4
cribed ty
, & Man.
» diet. ff
ccurred iy
of huma
ents wer
rt dietary

nan. Four

‘ole carbo.§ the Natl. Heart Inst. and the American Heart
laily for3§ Assoc.)
tolerant 34, Nature of visual pigment of the gecko.

were per-
3 on a diet

FREDERICK CRESCITELLI. Dept. of Zoology, Univ.
of California at Los Angeles, Los Angeles.

quantity Extracts of the retina of the Australian gecko,
e feeding Phyllurus milii, have revealed the occurrence of
travenou§ s unusual photosensitive pigment. The absorp-
nents, Wf tion spectrum of this pigment is characterized by
4s demon- amaximum, unusual for pigments from terrestrial
f fructoe§ nimals, at about 524 my. The pigment is not a
ose tolé-fnember of the porphyropsin system for the
that the product of bleaching appears to be retinene;.
ortal veitf The spectral sensitivity data of Denton obtained
a8 the till with Gecko gecko, which is in the same taxonomic
termining family as Phyllurus, fits the absorption curve of
iabetesitf the retinal pigment of Phyllurus. This pigment
eeded Wf is of special interest in view of the hypothesis of
this phe} Walls that the ancestral geckos were diurnal
animals with pure cone retinas and that a trans-
mutation of cones to rods has occurred in the
course of evolution. In the light of this hypothesis,
itmay be speculated that the present gecko pig-
ment is related to the ancestral cone pigment,
possibly even having had its origin from it. The
rsults with Phyllurus appear to be typical of
nocturnal geckos. Other species have been ex-
amined and all of them are characterized by the
presence in retinal extracts of this unusual pig-
ment in the general region of 520 mz. Rhodopsin
has, thus far, not been detected in any gecko.

hyper-
tid sinus
nD G. E.
Medicim,

rted that
nus ares,
al carotid
sulted in
f 10 dog
ly, these
onths and
. Genenl
perature,
1 normal.
mologous
» striking
ertensivt
nus ares
.V.) pr

15. Effectiveness of the visible spectrum
in controlling growth and differentiation
of fern prothallia. Witu1am J. Crotty
AND Sy.tv1A FRANK (introduced by Matvina
ScuwE1zER). Dept. of Biology, Washington Square
College, New York Univ., New York City.

This paper will describe preliminary studies on
ismall developing plant which may prove to be a
weful tool in separating out and defining cell

AMERICAN PHYSIOLOGICAL SOCIETY 43

division, cell differentiation and cell elongation.
The plant is the developing haploid spore of the
fern Polypodium aureum. The earlier literature
shows that when this plant is grown under con-
ditions of low illumination, it assumes a filament-
ous appearance. By altering the quality and
quantity of light, a reorientation of the cell
spindles occurs, and the resulting plant begins to
assume a plate-like appearance. Later the typical
heart-shaped form of a fern prothallium appears.
This effect of light on growth and form, moreover,
is reversible. That is, a plant having a plate-like
growth will, when transferred to light favoring
filamentous growth, begin to put out filaments.
And a filamentous plant can be induced to grow
into a plate-like form when the lighting condi-
tions are altered in the appropriate way. Thus,
light can be used as a tool to induce cell divisions
all in one plane, or to cause cell divisions to occur
in many directions. In addition, this light effect
can be used as a key to the underlying mechanism
controlling growth and form. An action spectrum
will be described that will relate the effectiveness
of various regions of the visible spectrum in
stimulating the reorientation of the cell spindles,
leading to plate-like growth. These results will be
interpreted in terms of the balance between
hormonal and nutritive factors in the plant.

136. Effect of environmental temperature
rhythms on blood and serum +olumes and
body water in dairy cattle. Homer E. DALz,*
SaMUEL Bropy AND Guiortia J. Burae*. Depis.
of Dairy Husbandry and Veterinary Physiology,
Div. of Agricultural Science, Univ. of Missouri,
Columbia.

Data were obtained on Jersey and Holstein cows
under 4 diurnally variable temperature rhythms:
10-40°, 40-70°, 60-110° and 70-100°F. Relative
humidity was roughly constant at 60%. The cows
were kept at each condition for about 3 wk., with
1 wk. rest at 60°F before the 70-100°F period.
Serum and blood volumes were estimated with the
T-1824 hematocrit method; body water by anti-
pyrine dilution. Blood and serum volumes were
20-30% higher during the high temperature
rhythms. Blood volumes of lactating Jerseys were
10% above those of dry Jerseys, of lactating
Holsteins 10-35% above lactating Jerseys and 20-
40% above dry Jerseys. Serum volumes were 20-
40% higher in lactating than in dry Jerseys,
40-50% higher in lactating Holsteins than in dry
Jerseys, and 10-20% higher in lactating Holsteins
than in lactating Jerseys. Absolute blood volumes
(ce/kg b. wt.) ranged from 63.3 to 105.7 in lactating
Holsteins, from 54.3 to 84.3 in lactating Jerseys
and from 47.7 to 88.0 in dry Jerseys. The serum
volumes ranged from 42.5 to 68.6 in lactating
Holsteins, from 33.2 to 56.6 in lactating Jerseys,

and from 28.3 to 47.1 in dry Jerseys. Hematocrits
ranged from 29.1 to 50.5% in dry Jerseys, from
27.6 to 40.2% in lactating Holsteins and Jerseys.
Antipyrine space did not seem to measure body
water as the volume of dilution ranged from 45 to
75% of body weight; serum water varied between
89 and 92%. (Supported by Atomic Energy Com-
mission.)

137. Pituitary-thyroid function in the epi-
nephrine-treated rat. 8. A. D’ANGELo. Jef-
ferson Med. College, Philadelphia, Pa.

The response of the pituitary-thyroid system of
adult female rats to epinephrine hydrochloride in
saline solution (1:1000) was determined in a series
of acute and chronic experiments. A single sub-
cutaneous injection of the hormone (50 ug/100 gm)
given 1-2 hr. prior to killing (24-hr. exp.) induced
a marked rise in BMR, decreased thyrotrophic
hormone (TSH) concentration in the adeno-
hypophysis, but failed to modify 24-hr. thyroidal
I'*| uptake or blood TSH levels. Administration of
epinephrine for 4-6 days decreased thyroidal
radioiodine collection and pituitary TSH yet did
not alter TSH titers in blood. The rate of bio-
logical decay of thyroidal I'*! was compared in
normal and epinephrine-treated rats (50 ug/100
gm daily, 28 days) by determining the reduction in
radioactivity over the gland utilizing an in vivo
detection technique (STEVENS AND D’ANGELO.
Proc. Soc. Exper. Biol. & Med. 85: 633, 1954).
Counts taken over a 96-120 hr. period during the
first and last week of the experiment revealed no
significant change in the decay rate of thyroidal
radioiodine. Biological half-lives with epinephrine
were 3.3 and 3.8 days, with 3.5 and 3.6 days the
values in saline-injected controls. The results lend
no support to the thesis that adrenal medullary
hormone activates the pituitary-thyroid system
of the rat. (Supported by funds from Grant
A-432, Natl. Insts. of Health, PHS.)

138. Inorganic phosphate and gastric acid
secretion in vitro. Horace W. DAvENpPoRT.
Dept. of Physiology, Univ. of Utah, Salt Lake
City.

Rates of acid secretion by mouse stomachs were
measured in vitro in standard phosphate-buffered
salt solution containing phosphate at 17.2 mm, in
glycine-buffered, phosphate-free solutions and in
solutions of intermediate phosphate concentra-
tions. In absence of stimulation by carbachol,
variations in phosphate concentration had little
or no effect upon acid secretion and oxygen con-
sumption. Under maximal stimulation by car-
bachol, optimal acid secretion at a rate about 20%
greater than that in the absence of inorganic
phosphate occurred at a phosphate concentration
of 8 mm. Inorganic phosphate was lost to the
bathing solution by stomachs incubated in phos-

FEDERATION PROCEEDINGS

Volume 1;

phate-free glycine buffer. The rate of loss was
rapid in the first 10 min., but thereafter it was
smaller and steady for the remainder of the hour
period of incubation. Stimulation by carbachol
increased the rate of loss in the first 10 min. but
had no effect upon the subsequent rate. Dinitro.
phenol in concentrations from 0.001 to 0.1 ma had
no effect upon the rate of loss of phosphate al-
though the latter concentration inhibits acid
secretion. The amount of inorganic phosphate logt
to the glycine buffer was five or more times greater
on the serosal than on the mucosal side so that,
despite a larger volume of solution on the serogal
side, the concentration of phosphate on that side
was always greater than on the mucosal side,
There appears to be no net transport of phosphate
from one side of the stomach to the other.

139. Erythropoietic activity in the burned
dog. Aupray K. Davis* anp Epwarp L,
ALPEN. U. S. Naval Radiological Defense Lab.,
San Francisco, Calif.

Previous studies from this laboratory have
characterized the anemia of thermal injury in the
rat as one of rapid hemolytic removal of cells
accompanying increased rates of erythrocyte pro-
duction. However, studies from other laboratories
have led to conclusions of existing dishemato-
poiesis in the dog. We have therefore also ex-
tended our earlier studies to the dog wherein
infection may have a major influence on hemato-
poiesis. At various times during the recovery
phase from a 15% body area full thickness burn
the plasma iron turnover rate, erythrocyte ‘up-
take’ curve and red cell volume were measured.
The red cell volume and hematocrit fell pro-
gressively during the first 2 wk. and remained at
these new low levels 30-45 days. The plasma turn-
over rate was unchanged or slightly elevated;
however, the plasma inorganic iron pool was
markedly smaller. This lowering of serum iron
was accompanied by a compensating increase in
slope of the Fe® plasma disappearance curve that
maintained the turnover rate (in mg Fe/day) in
the normal range. Curves of appearance of radio-
activity in erythrocytes, instead of showing the
normal increase with plateauing at 5-7 days, show
an abortive or minimal rise followed by a rapid
disappearance of radioactivity that could only be
attributed to rapid destruction of erythrocytes.
Burn anemia in the dog is characterized as 8
hemolytic process accompanied by near normal
rate of erythropoiesis. The failure of ‘uptake
curves’ as a measure of function in the presence
of rapid destruction is obvious in these studies.

140. Effects of drugs on locomotor activity of
monkeys. GrorcE D. Davis (introduced by
Leon CuurneEy). Dept. of Physiology, Louisiana
State Univ., New Orleans.

March

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March 1956

Control measurements of locomotor activity
were made in normal monkeys and in monkeys
which had been rendered hyperactive by unilateral
or bilateral lesions of the caudate nucleus. These
animals were then given various dosages of
reserpine (Serpasil) and methyl-phenidylacetate
Ritalin) and locomotor activity tests were re-
peated. In both the normal and the hyperactive
animals, a clear diminution of spontaneous
activity was seen following administration of as
little as 0.1 mg/kg reserpine per day for several
days. In all animals tested, 0.25 mg/kg/day of
reserpine was sufficient to eliminate virtually all
spontaneous activity for several hours following
administration. Equal doses of reserpine pro-
duced an equivalent activity reduction per-
sentagewise in normal animals with little spon-
taneous activity and in animals having large
caudate lesions with a consequent extreme hyper-
activity. The characteristic activity pattern of
the individual animals was not affected by reser-
pine. With the use of methyl-phenidylacetate, the
expected increase in the activity of the isolated
monkey was not observed. Dosages as large as
5 mg/kg were employed. Although the animals
showed considerable agitation in the normal
living quarters and during the transfer process to
and from the test cage, they remained at previ-
ously measured levels of activity during the
testing period. Some minor changes of behavioral
patterns were seen during these tests. The effect
of methyl-phenidylacetate would thus appear to
be that of increasing the reaction of the animal
to external stimuli. (Supported in part by re-
search Grant B-874 from the NINDB, PHS.)

Ml. Effect of volume and rate of inflation on
transpulmonary pressure and response of
pulmonary stretch receptors. Harry L.
Davis,* Warp §S. FowierR anp Epwarp H.
LaMBERT. Mayo Clinic and Mayo Fndn., Roches-
ter, Minn.

Action potentials of 53 single vagal afferent
nerve fibers from pulmonary stretch receptors,
transpulmonary pressure and rate of air flow were
recorded in 16 anesthetized cats with open thor-
axes during varied volumes and rates of inflation.
The large majority of receptors were slowly
adapting and were located peripheral to the
trachea and extrapulmonary bronchi. Discharge
frequency varied approximately linearly with the
volume of inflation at static volumes and during
slow inflation. During rapid inflation, discharge
frequency and transpulmonary pressure were in-
creased over values observed at similar static
volumes. Transient discharges frequently could
be produced during small but rapid inflations,
even though the volume added was so small that
the receptor remained below its static volume

AMERICAN PHYSIOLOGICAL SOCIETY 45

threshold at the end of flow. At a given volume,
discharge frequency was usually less during de-
flation than during inflation. Both discharge
frequency and total transpulmonary pressure
varv with lung volume and its rate and sign of
change. The results suggest that the receptors are
located in the peripheral bronchi or bronchioles
and have bearing on the work of breathing and its
regulation.

142. Adrenergic activity of canine vascular
bed. C. P. Drat, Jr.,* Harotp H. CHaKaLes*
AND Haro.p D. Green. Dept. of Physiology and
Pharmacology, Bowman Gray School of Medicine,
Winston-Salem, N.C.

Hepatic arterial and portal venous blood flows
(electromagnetic flowmeter) and pressures were
recorded in the pentobarbitalized dog. Intra-
arterial injections of l-epinephrine (EPI) and 1-ar-
terenol (ART) caused a small reduction in portal
flow and increase in portal pressure and resistance
and a larger decreased flow and increase in re-
sistance in the hepatic artery. Injections into the
vein caused a prompt increase in portal pressure
and resistance and a small decrease in flow but
were almost without effect on the arterial bed.
In both, the changes were more marked with EPI
than with ART. Isopropyl-arterenol (ISO) given
in either the portal vein or hepatic artery caused
a small delayed increase in hepatic artery resist-
ance and decrease in portal resistance. Nerve
stimulation (SPL) caused an increase in portal
pressure and resistance and a small rise, then fall,
in portal flow but had little effect on the arterial
bed. Small doses of azapetine abolished the effect
of ART and SPL; larger doses abolished the effects
of EPI. Reversal of the constrictor response to
EPI was not seen with any dose. It was concluded
that hepatic arterioles are much more responsive
to EPI than to sympathetic stimulation or ar-
terenol; they are inaccessible to substances arriv-
ing via the portal vein. Hepatic ‘venules’ are more
responsive to EPI, ART and SPL than are arteri-
oles and are accessible to substances arriving by
way of both vein and artery. No adrenergic dilator
receptors were found in either venules or arteri-
oles. (Supported by Public Health Service Grant
H-487.)

143. Attraction and avoidance evoked by sep-
tal and rhinencephalic stimulation in the
monkey. Jose M. R. Detaapo anp Ben Bur-
sTen.* Depts. of Physiology and Psychiatry,
Yale Univ. School of Medicine, New Haven,
Conn.

In a group of 6 monkeys permanent multilead
electrodes were implanted in septum pellucidum,
amygdala, fornix and other cerebral structures.
By means of a belt and a chain each animal was

tested for 1-2 hr. daily on a rectangular table
around which it could move freely. The points of
the brain tested at random were electrically
stimulated only when the monkey was on one side
of the table. The sides were altered randomly.
Excitation consisted of unidirectional rectangular
pulses, 100 cps, 0.24 msec. of pulse duration,
0.8-1.0 ma, applied for 0.3 sec. every 4 sec. Early
in the testing, the monkey was compelled to sam-
ple both sides of the table. In each animal elec-
trical stimulation of some specific points of septum
or amygdala increased the time the animal spent
on the stimulated side. The statistical significance
of the results was of the order of 1-5% level of
confidence. Motor activity of the animals was not
affected during the stimulations, nor were there
gross disturbances of the electrical activity of the
brain. Stimulation of one septal point in 2 mon-
keys and of one amygdala point in another mon-
key decreased the time the animals spent on the
stimulated side. Electrical stimulation of other
points did not modify the time the animal spent
on either side of the testing table. It may be con-
cluded that stimulation of some cerebral areas is
attractive, while the stimulation of other points is
unattractive for the animal. Neutral areas also
exist. (Aided by grants from the Foundations’
Fund for Research in Psychiatry and ONR, Nr
113-320.)

144. Body motility during sleep and dream-
duration recall. WiLLiIAM DEMENT* AND
NATHANIEL KierTMAN. Dept. of Physiology,
Univ. of Chicago, Chicago, Ill.

As previously reported (Federation Proc. 14:
216, 1955), during sleep the EEG exhibits a series
of successive variations, with discrete periods of
rapid eye movements occurring during the ‘light-
est’ phase of each cycle. Body motility in 20 and
dream recall in 2 subjects were studied in relation
to these variations. Composite histograms re-
vealed that body motility 1) gradually decreased
as sleep ‘deepened’ and then rose to a peak with
the lightening of the EEG, decreasing again as
the next cycle started, and 2) dropped sharply
at the onset of the rapid eye movements, rebound-
ing abruptly as the eye movements ceased. One
subjeet gave a detailed description of dream con-
tent in 36 of 40 awakenings while the eyes were
moving, with a general correspondence between
the length of the reports and duration of the eye
movement periods. No dream recall was elicited
after 26 awakenings at other times. The subject
was awakened 21 times either 5 or 15 min. after the
eye movements (dreaming) had begun and judged
the dream duration correctly each time. Findings
in the second subject confirmed those of the first.
(Work done under a Public Health Service Fellow-
ship and aided by a grant from the Dr. Wallace C.

FEDERATION PROCEEDINGS

Volume if

and Clara A. Abbott Memorial Fund of the Uniy.
of Chicago.)

145. Adrenergic drugs and blockade on coro.
nary blood flow. Apam B. DEnison, Jr,
SutHip BARDHANABAEDYA* AND Haroxp J,
GREEN. Dept. of Physiology and Pharmacology,
Bowman Gray School of Medicine, Winston.
Salem, N.C.

The myocardial and coronary vasomotor re-
sponses to intracoronary epinephrine, artereno|
and isopropylarterenol were studied in open-
chested, pentobarbitalized dogs during varioug
degrees of adrenergic blockade produced by intra-
arterial Azapetine. The heart was exposed in the
customary pericardial sling and the left coronary
artery cannulated and perfused with blood from
the left carotid; phasic flow and pressure were re-
corded by an electromagnetic flowmeter and a
Statham pressure transducer on a Sanborn Poly-
viso. All 3 adrenergic drugs in 1 wg doses increased
approximately equally the vigor of myocardial
contraction, evidenced by backflow during igo-
metric contraction and by increased systolic
peripheral coronary pressure (perfusion supply
temporarily occluded). All 3 drugs produced ap-
proximately equal vasodilation, shown by in-
creased diastolic flow. The magnitudes of the
above effects increased with the 3 and 10 ug doses.
None of the drugs caused vasoconstriction. Vaso-
dilation preceded the myocardial response to
isopropylarterenol; 3 and 10 ug of arterenol caused
frequent ventricular premature beats. One milli-
gram of Azapetine had no noticeable effect on the
adrenergic responses; 3-30 mg produced marked
ECG changes, bradycardia, unstable arterial
pressure and often ventricular fibrillation but
caused only a slight reduction of the response to
the adrenergic drugs. Thirty to 100 mg of Azape-
tine blocked the myocardial stimulant responses;
100 mg or more was required to block the vasomo-
tor responses. (Supported by Public Health Serv-
ice H-1094.)

146. Factors involved in intact vessel electro-
magnetic flow recording. ADAM B. DENISON,
JR. AND MERRILL P. Spencer. Dept. of Physiol-
ogy and Pharmacology, Bowman Gray School of
Medicine, Winston-Salem, N. C.

The electromagnetic flow recording principle,
while quite simple in theory, has been difficult to
apply in practice because of the ease with which
artifacts could obscure the results; especially is
this true of recording from intact vessels. This
derives largely from the fact that the recording
electrodes receive not only the flow signal but also
superimposed disturbances from the magnet and/
or electrode contact potentials and other inter-
ference such as ECGs, AC hum, etc. Use of 4

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March 1956

square-wave AC magnet limits the duration of the
magnet artifact, leaving quiet periods during
which the flow is recorded. Progressive increase
of the carrier frequency to the present 240 cps
has minimized interference from ECGs and AC,
and also permits high frequency phasic recording.
Artifacts may arise from intermittent metallic
contacts such as hemostats rubbing together with
respiratory movements; this may be prevented by
inserting a gauze sponge between such instru-
ments. Interposing the vessel wall between the
signal and the electrodes reduces the sensitivity
considerably, although this reduction is quite con-
stant with different vessels. The vessel should be
restricted in one diameter so that it can bulge
only in the diameter being recorded. Immersing
the vessel and pickup unit in saline reduces the
calibration sensitivity somewhat; if the vessel
fits the pickup satisfactorily it is sufficient to keep
it moist without submerging it entirely. Variations
in hematocrit alter the calibration in a linear
manner. Satisfactory and reproducible results
can easily be obtained if these various factors are
borne in mind during the routine calibration and
application of such equipment. (Supported by
Public Health Service Grant H-1094.)

147. Glucuronidase activity in liver and kid-
ney from animals exposed to a low environ-
mental temperature. M. E. DENISON AND R.
L. JASPER (introduced by W. A. Hrestanp).
Depts. of Physiology and Environmental Medicine,
Army Med. Research Lab., Fort Knox, Ky.
The present study was undertaken to further

investigate the sexual dimorphism in survival

previously observed during exposure of rats to a

low environmental temperature. In view of the

differences in kidney tissue metabolism of intact
and castrated male rats exposed to cold and of the
differences in glucuronidase activity in intact and
castrated male rats it was thought worthwhile to
study the glucuronidase activity of cold exposed
rats. Castration increases the glucuronidase activ-
ity of liver slices from rats kept at room tempera-
ture. Cold exposure of male rats and of castrated
male rats tends to decrease the liver glucuronidase
activity. Castration results in a decrease in renal
glucuronidase activity of the order of 20%. Kidney
glucuronidase activity is decreased in rats exposed
to a low environmental temperature. This de-
crease is approximately 24% in intact male rats
and approximately 40% in castrated rats. Treat-
ment of intact and castrated male rats with sesame
oil at room temperature does not significantly
alter the kidney glucuronidase activity. Treat-
ment with testosterone propionate tends to in-
crease the renal glucuronidase activity. It is
postulated that cold and the level of both corti-
sone and testosterone propionate is influencing

AMERICAN PHYSIOLOGICAL SOCIETY 47

the glucuronidase activity in the kidney in, as yet,
some unexplained manner.

148. Active transport of sodium ion in the
nonacid secreting stomach. WarrREN H.
Dennis,* ALAN M. BorNSTEIN* AND WARREN
S. Reum. Dept. of Physiology, Univ. of Louis-
ville School of Medicine, Louisville, Ky.

It has been previously shown that in the non-
acid secreting stomach there is a net transport
of chloride ion from the interstitial fluid to a
0.05 m NaCl solution in contact with the mucosal
surface. Since this represents transport against
its electrochemical potential gradient (ECPG)
it can be defined as active transport. With 0.20
M NaCl bathing the mucosa the Cl- ion is trans-
ported in the direction of its ECPG. In the pres-
ent experiments the chambered gastric segment
preparation (anesthetized dogs) was used and the
Cl- ion, Nat ion (Zn uranyl acetate method),
volume and freezing point changes of mucosal
fluid and the p.d. across the stomach were meas-
ured. It was found that the Na* ion was trans-
ported in the direction of its ECPG (ISF to lumen)
when 0.05 m NaCl was the mucosal fluid. With
0.20 m NaCl as the mucosal fluid it was found that
the Na* ion was transported against its ECPG
(lumen to ISF). With 0.20 m NaCl there was a
consistent net increase in the volume of mucosal
fluid so that the Nat ion was transported against
the net water drag. The data with 0.20 m NaCl
solutions are interpreted as indicating an active
transport of Nat ion in the resting stomach from
lumen to ISF.

149. Multiple components of single motor
units. J. S. Denstow, M. O. GuTEeNsoun,* J.
A. Cuace* anp M. G. Kumm.* BioMechanics
Lab., Kirksville College of Osteopathy and Surgery,
Kirksville, Mo.

Single motor unit action potentials have been
described by many investigators as being mono-,
di-, tri- or polyphasic. In these studies, made
with multiple electrode placements in certain
muscles of the cat and human, it has been shown
that a) while at one electrode placement the action
potential may be mono-, di-, tri-, or polyphasic,
this simply represents unit activity at that loca-
tion, and b) a composite of the action potentials
from each single unit, as recorded from multiple
electrode placements, is always polyphasic. The
polyphasicity may be due in part to propagation
delay because the action potential passes different
electrodes at different times. However, since a
fortuitous placement of a single electrode permits
the recording of an extremely complex action po-
tential from a single unit at one location, it is
apparent that the polyphasic action potentials of
single units are due in part to temporal dispersal

48 FEDERATION PROCEEDINGS

of the activity of the different components of
single units. The possibilities that polyphasic
action potentials from a single unit might be due
to a) double discharges of one unit or b) the tem-
porary synchronization of two units have been
eliminated. It has been shown that single motor
units are made up of multiple muscle components
arranged in a strip-like pattern. These components
fire asynchronously; however, each component
retains the same time relationships to the other
components every time the unit is activated.
(Supported by grants from the Natl. Heart Inst.
and the American Osteopathic Assn.)

150. Metabolic response of warm and cold
acclimated rats to very cold environments.
FLORENT Depocas (introduced by E. Page).
Div. of Applied Biology, Natl. Research Labs.,
Ottawa, Canada.

The heat production (indirect) of 30°C accli-
mated (J) and 6°C acclimated (JJ) Sprague-
Dawley adult male rats has been determined
with an open circuit metabolism apparatus over
the temperature range —36°C to 30°C after an
equilibration period of 20 min. or more. At all
temperatures heat production of JJ was higher
that that of J, but, the slope of the heat production
vs. temperature curve was similar in both groups
at least between —7°C and 20°C. At lower tem-
peratures, the heat production of J fell with time
of exposure to the test environment so that the
open circuit apparatus due to its lag was useless
for determining true maximal metabolic rate. A
constant volume closed circuit metabolic system
with a lag of only 2.5 min. was devised and used
for measurements of the average heat production
of J and JJ at —25°C between the 3rd and 20th min.
of exposure. The results indicate that 1) a rise in
heat production of 2.5 and 3 times basal takes
place in both groups of rats within 3 min. after
exposure and is apparently maintained until the
end of the 20 min. test. 2) The value given by IJ
is the one expected from the slope of the heat
production curve at higher temperature while
that given by J is lower than that expected. In
summary, the metabolic response of white rats
to cold exposure is extremely rapid and at the
lower temperatures J reaches a maximum meta-
bolic rate while J7 can still show an increase.

151. Renal metabolism of 5-hydroxyindole
acetic acid. AGAMEMNON DESPOPOULOS (in-
troduced by E. M. Renxin). National Heart
Institute, Bethesda, Md.

The ability of slices of kidney cortex to concen-
trate certain of the organic acids which are ex-
creted by renal tubular activity has served as the
basis for an in vitro technique for the study of
renal tubular excretory activity. The data are

Volume ig

expressed as an S/M ratio which defines the ratio
of the concentration of the test substance in eagh
gram of tissue to that in each cubic centimeter of
medium at the end of the incubation period. 5.
hydroxyindole acetic acid (5-HIAA) was exam.
ined by this technique and the data were com.
pared with those for p-aminohippuric acid (PAH),
a compound conceded to be excreted by tubular
activity. In oxygen, both 5-HIAA and PAH were
accumulated by rabbit kidney slices, the §/M
values being 5.4 and 10.0, respectively. In nitro.
gen, the S/M ratio for each compound was re.
duced to one, indicating a cessation of the active
accumulation process. For each compound, the
S/M ratio was increased by acetate and was re.
duced by 2,4-dinitrophenol and phenol red. The
S/M PAH was depressed by 5-HIAA (5 X 10-4y),
but PAH (5 X 10-‘m) did not depress the §/M
5-HIAA. When 5-HIAA was concentrated in the
slices, only 70% could be recovered from the com-
plete system. This loss of material was not ob-
served in an atmosphere of nitrogen. The intraye-
nous administration of 2 gm of probenecid to
patients with a high rate of excretion of 5-HIAA
was followed by a decrease in the rate of excretion
of 5-HIAA and an increase in its concentration in
the plasma. These data suggest that 5-HIAA and
PAH are excreted similarly by the kidney by
glomerular filtration and tubular excretion.

152. Central pathways subserving weight dis-
crimination in monkey. JuNE L. DEVrto*
AND THEODORE C. Rucu. Dept. of Physiology and
Biophysics, Univ. of Washington School of Medi-
cine, Seattle.

Monkeys were trained to discriminate weights
by the pull-in method, and threshold curves ob-
tained pre- and postoperatively with progressively
decreasing weight differences. Complete section
of the dorsal columns at the level of cervical seg-
ments C2-C3 caused a transient loss in weight dis-
criminatory ability with recovery to preoperative
levels upon retraining. Although bilateral antero-
lateral cordotomy alone did not impair discrimi-
nation, addition of dorsal column lesions produced
a severe deficit, suggesting an alternate sensory
pathway in anterior columns. Further, parietal
lobectomy secondary to dorsal column lesions
caused a persistent deficit in weight discrimina-
tion, indicating that the fibers responsible for
recovery of function after dorsal column section
project in part to parietal cortex. The existence of
an afferent pathway from deep nerves to parietal
cortex, independent of the dorsal column relay
system, was confirmed by electrical recording
methods. In monkeys and cats, primary evoked
potentials were recorded from primary somato-
sensory areas after complete interruption of the
cervical dorsal columns. Spinal lesions in monkeys

Mar

were
ton

ume I§

e ratio
iD each
eter of
iod. 5.
exam.
> com-
PAR),
ubular
I were
e S/M
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d, the
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d. The
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-HIAA
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it dis-
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eights
es ob-
ssively
ection
al seg-
ht dis-
srative
ntero-
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ensory
arietal
lesions
imina-
le for
ection
nce of
arietal

relay
ording
voked
ymato-
of the
onkeys

March 1956

were checked “histologically. (Aided by Washing-
ton State Fund for Biology and Medicine.)

133. Shifts of water and electrolyte content of
rat renal cortical tissue in simple solutions
in vitro. INcritH Dryrup. Depts. of Zoology,
Barnard College and Columbia Univ., New York
City.

In an attempt to analyze further the demon-
strated reversal of fluid uptake by rat kidney cor-
tical slices immersed in relatively simple solutions
approximating rat plasma in tonicity (300 mOs/1.),
observations have been made on Na, K and water
content of renal tissue under a variety of condi-
tions. Of particular interest is the observation
that tissues, hyperhydrated by immersion under
metabolically unfavorable conditions in 0.15 m
NaCl, subsequently lost large amounts of Na,
without significant change in K content, paral-
ling water loss during incubation in 0.15 m
NaCl at 37°C, with oxygen atmosphere. Results
of experiments in which renal tissue was incubated
in other simple solutions have been compared with
the findings on immersion and incubation in 0.15
u NaCl. (Part of this work was carried out in
Denmark in the laboratory of Dr. Hans Ussing,
and part in New York with support from research
gant #H-2061, from the Natl. Heart Inst.,
PHS, and from the Higgins Fund.)

14. Fibrinolytic, coagulant and hemolytic
properties of certain snake venoms. PavuL
DipISHEIM* AND Jessica H. Lewis. Dept. of
Medicine, Univ. of Pittsburgh School of Medi-
cine, Pittsburgh, Pa.

In a search for an ideal in vivo clot-dissolving
agent, crude preparations of certain snake ven-
oms, and several other proteinases and activators,
were examined in vitro. Each substance was
tested for its ability to dissolve human fibrinogen,
formed fibrin clots and whole blood clots. In
addition, each was tested for thrombic, antithrom-
bic, thromboplastic, anticoagulant and hemolytic
activities. All venoms studied showed activity
in one or more of these test systems. Seven ven-
oms (B. neuwiedii, B. alternatus, B. jararaca, B.
aroz, C. horridus, C. adamantus, V. russelii) were
found to be thrombic, i.e. they clotted human
fibrinogen or plasma and were also fibrinolytic.
Four venoms (A. piscovorus, C’. atrox, A. mokasen,
¢. basilicus) were fibrinolytic and anticoagulant
without being thrombic. The anticoagulant action
was due to fibrinogenolysis. Other venoms tested
(C. terrificus, crude cobra) were not actively fibrin-
dlytic but both were hemolytic. Three venoms
(B. atroxz, C. horridus, C. adamantus) were as
thromboplastic as a commercial preparation of
Russel’s Viper Venom. All 8 venoms which de-
stroyed fibrinogen were hemolytic, hence could

AMERICAN PHYSIOLOGICAL SOCIETY 49

probably never be of clinical use. Trypsin, in
contrast, was fibrinogenolytic without being
hemolytic in the concentrations used. A crude
factor was prepared from normal human urine
which lysed fibrin and whole blood clots but was
also hemolytic. Streptokinase and staphyloki-
nase, acting as activators of the plasminogen
present in the clots, were fibrinolytic without
being hemolytic or exhibiting any appreciable
detrimental effect on the remainder of the coagu-
lation mechanism as tested.

155. Physiological disposition of Dilantin
(diphenylhydantoin) in animals and man.
W. A. Diuu,* A. Kazenxo,* L. M. WoiF* anp
A. J. GuazKo. Research Labs., Parke, Davis and
Co., Detroit, Mich.

Concentrations of Dilantin in biological mate-
rials were determined by a new colorimetric pro-
cedure involving double extraction into chloro-
form, evaporation of the solvent, nitration of the
residue, reduction with stannous chloride, diazo-
tization and coupling with the Bratton-Marshall
reagent. Oral administration of Dilantin to rats
and dogs in doses of 100 mg/kg produced maxi-
mum serum levels of 8 y/ml in 8-12 hr., with a re-
turn to zero levels in 30-40 hr. Tissue levels gen-
erally ran parallel to blood levels. Concentrations
of Dilantin in liver were 3-fold greater than in
serum, with progressively lower levels being found
in brain, heart, lung, kidney, spleen and skeletal
muscle. Unchanged Dilantin was isolated from
dog and rat liver by countercurrent extraction
procedures and identified by its infrared absorp-
tion spectrum. Less than 1% of the dose was ac-
counted for by urinary excretion, but Dilantin
was also found to be excreted in the bile. The
nature of the metabolic products of Dilantin is
now under investigation with C4-labeled material.
Administration of single oral doses of Dilantin
(400 mg sodium salt) to 6 normal adult human
subjects produced maximum blood serum levels
of 2 to 5 y/ml in 8-12 hr. The blood levels were
found to drop off at the rate of 50%/17-24 hr. A
few subjects showed levels greater than 2 +/ml
48 hr. after dosage.

156. Effect of choline deficiency on reticulo-
endothelial system of the rat. N. R. Di
Luzio (introduced by R. H. AupEn). Div. of
Physiology, Univ. of Tennessee, Memphis.

It has previously been demonstrated that the
lipotropic agent, choline, stimulates the activity
of the reticuloendothelial system (RES). Since
various procedures, such as X-irradiation and cor-
tisone administration produce hyperlipemia,
fatty livers and, simultaneously, a depression in
RE function, a correlation between the activity
of the RES and lipide metabolism is suggested.

50 FEDERATION PROCEEDINGS

The present study was undertaken to determine
whether the fatty liver degeneration which occurs
in the choline-deficient rat is related to an altera-
tion in the functional activity of the RES. Radio-
active colloidal gold was employed to measure the
phagocytic velocity, capacity and organ uptake
in normal and choline-deficient rats. The capacity
of the RES was determined by the administration
of Thorotrast to both groups. In the choline-
deficient rats, no alteration was found in the rate
of removal of Au'® from the vascular system

Spleen and liver uptake did not deviate from
normal, even though the liver cholesterol and
triglyceride was increased 2- and 5-fold, respec-
tively. Lung, however, showed a 5-fold increase
in radioactivity. The injection of Thorotrast prior
to Au!®, produced a 3-fold decrease in the vascu-
lar clearance of Au! in the normal rats, although
the ultimate tissue distribution of the colloid was
unaffected. In marked contrast, Thorotrast ad-
ministration to the choline-deficient rats produced
no alteration in phagocytic velocity, indicating
that a marked increase in the capacity of the RES
occurred in those animals which had severe fatty
livers. (Supported in part by a grant from the
Atomic Energy Commission.)

157. Role of the kidney in etiology of renal
hyperlipemia. N. R. D1 Luzio* anp C. RrLey
Hovwck.! Div. of Physiology, Univ. of Tennessee,
Memphis.

The development of a hyperlipemia after bi-
lateral nephrectomy or ureter ligation has led to
the hypothesis that the kidney exerts a regulatory
influence on plasma lipides. This study of the
plasma lipide partition in bilaterally nephrec-
tomized dogs, where excretory function was main-
tained by intermittent peritoneal dialysis, was
carried out to determine more precisely the role
of the kidney in the etiology of renal hyperlipe-
mia. The concentration of plasma phospholipide,
cholesterol, triglyceride and nonprotein nitrogen
was determined in 7 dogs prior to, and at the 7th
and 14th day, after bilateral nephrectomy. At
these times, no significant alteration in the con-
centration of phospholipide and cholesterol was
observed. The triglyceride concentration was sig-
nificantly elevated 40% over the control value at
the 7th and 14th day. At no time was a physical
lipemia observed in these animals. The nonprotein
nitrogen level was increased approximately 5-fold
at that 7th and 14th day. These findings, of essen-
tially unaltered lipide concentrations in main-
tained bilaterally nephrectomized dogs, is in
marked contrast to evidence previously reported
by other investigators who demonstrated pro-
gressive and continuous increases in plasma lipides

1 Deceased.

Volume ij

after bilateral nephrectomy or ureter ligation jp
many species. Apparently, the renal hyperlipemig
previously reported resulted from a metabolie
disturbance due to the loss of renal excretory
capacity and not a loss in any renal hormonal
or metabolic function as has been previously
postulated.

158. Clearing of neutral fat by tissue lipase,
Renatp R. DiNeLia AnD H. C. MENG (intro.
duced by Extiot V. Newman). Dept. of Physi-
ology, Vanderbilt Univ. Med. School, Nashville,
Tenn.

A method for the purification of an enzyme in
duodenal mucosa that clears neutral fat has been
developed. Intestinal mucosa from hogs wag
homogenized in a Waring Blendor with 4 parts
H.O (0°). Following centrifugation at 2,500 X 9,
an acetone powder of the supernatant was pre-
pared at room temperature with 10 volumes of
acetone. The powder was extracted at room tem-
perature with phosphate buffer (0.05 m, px 6.0).
The extract was heated for 4 min. at 55° to remove
inert proteins. Mucin was precipitated with acetic
acid (1%) at —5°. The supernatant was immedi-
ately adjusted to pu 6.7 and ethanol was slowly
added to a concentration of 28%. Approximately
50% of the precipitate obtained after centrifuga-
tion was soluble in phosphate buffer (0.067 x,
pH 7.6); the remaining insoluble material was dis-
carded. The redissolved ethanol fraction was
brought to 55% saturation with solid ammonium
sulfate. The activity of the precipitate was de-
termined. This procedure gave a 50-fold increase
in specific activity, with a yield of 6%. The enzyme
was nondialyzable, PCMB sensitive, and was in-
activated at 65°. It was characterizable as a lipase
on the basis of 1) quinine sensitivity, 2) accelera-
tion by bile salts and Catt, and 8) resistance to
E600 (10 m) eserine, and atoxyl. The enzyme
could be extracted from the intact cellular sub-
particles by either serum or alkaline buffers. The
alkaline extract was inhibited by serum albumin,
higher concentrations of heparin and was prota-
mine insensitive. In contrast to steapsin, the
mucosal lipase was active toward monoglycerides
and hydrolyzed mono-, di-, and triglycerides at
identical rates.

159. Refractory and latent periods of prema-
ture ventricular systole. JosEPH R. D1PA.MA.
Dept. of Pharmacology, Hahnemann Med. Col-
lege, Philadelphia, Pa.

Electrodes were placed on the atrium and ven-
tricle of anesthetized open-chest dogs. After
crushing the sinoatrial node the heart was driven
at a rate of from 90-180/min. by the first pulse from
a special stimulator. The second pulse in each
cycle was delivered to the right ventricle so as to

lume 1%

tion in
lipemia
tabolie
pretory
rmonal
viously

ipase,
(intro-
Physi-
shville,

yme in
s been
S was
- parts
0 X 4,
1S pre-
nes of
n tem-
H 6.0).
emove
acetic
medi-
slowly
nately
‘ifuga-
)67 M,
as dis-
n was
onium
as de-
crease
nzyme
‘as in-
lipase
elera-
nce to
izyme
r sub-
3. The
umin,
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1, the
erides
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After
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-as to

March 1956

produce a premature ventricular systole. In this
manner the refractory period and the latent period
of the normal beat could be determined for the
particular strength of stimulus used. A third
stimulus in each cycle was also delivered to the
right ventricle at such a time as to produce a sec-
ond ventricular premature systole. This permitted
the estimation of the refractory period and latent
period of the first premature systole. It was found
that regardless of the heart rate the premature
yentricular systole always had a refractory period
and latent period much shorter than the preceding
normal systole. In other experiments it was pos-
sible to show that successive premature systoles
could be initiated at progressively shorter in-
tervals. It was also found that when the heart was
driven from a ventricular site the refractory period
and latent periods were decreased as compared to
the same heart driven from the atrium. The sig-
nificance of these results will be discussed. (Sup-
ported by grant #*H1508 from the Natl. Insts.
of Health, PHS.)

160. Extra-splanchniec blood volume: its im-
portance in calculation of liver blood flow
from colloid disappearance rate. ERNEsT L.
Dosson, GeorGE F. WaARNER,* CAROLINE R.
FinNEY* AND Dorotuy M. Apamson*. Donner
Lab., Univ. of California, Berkeley.

In previous communications, from this and other
laboratories, the liver blood flow has been calcu-
lated by multiplying the colloid disappearance
rate constant, k (from the equation C = Co e-*t)
by the total blood volume. Actually the proper
volume to use in this calculation is the volume of
blood which lies outside the splanchnic area. The
reason for this is that the event which leads to the
removal of a particle from possible detection in
the peripheral blood occurs when the particle
leaves the aorta destined for the liver rather than
when the particle is actually phagocytized by a
Kupffer cell. Since chromic phosphate does not
‘mix’ with the splanchnic blood, the ‘extra-
splanchnic’ blood volume can be estimated di-
rectly by extrapolating the chromic phosphate
disappearance curve, with certain corrections,
such as a zero time shift and a first pass elimination
fraction. Or, this ‘extra-splanchnic’ blood volume
can be obtained indirectly by subtracting the
splanchnic blood volume from the total blood vol-
ume. The time function of the ratio of colloid con-
centration to labeled albumin concentration will
hot change until blood which has been once
through the liver arrives at the sampling site.
Thus, by making a simple correction for injection
site to sampling site transit time, an estimate of
splanchnic circulation time can be obtained. If
uniform wash out of the splanchnic vascular bed
is assumed, its volume can be calculated by mul-

AMERICAN PHYSIOLOGICAL SOCILTY 51

tiplying splanchnic blood flow by splanchnic
circulation time.

161. Human stress response in contrasting
aircraft operations. THAappEUS J. DoMANSKI
(introduced by H. M. Sweeney). Dept. of
Pathology, USAF School of Aviation Medicine,
Randolph Air Force Base, Randolph Field, Texas.
Training missions flown in B-47 and B-29 type

aircraft were studied with respect to the incidence

of strain in student aircraft commanders and in
instructor pilots. The criterion of strain was the
occurrence of a pre- to postflight eosinopenia. On
this basis the incidence of strain for transition
missions was: a) 60% for B-47 instructor pilots,

b) 61% for B-47 student aircraft commanders, c)

22% for B-29 instructor pilots and d) 28% for B-29

student aircraft commanders. The contrast be-

tween B-47 and B-29 subjects was statistically
significant (P < 0.01). The eosinophil response
findings were in accord with the evaluation of
senior flying personnel as to the relative dif-
ficulty of the missions studied. In the study of
instructor-student pairs, it was found that the
incidence of strain for student aircraft com-
manders was higher but not significantly different
from that of the corresponding instructor pilots.

162. Chemical and nutritional recovery from
an x-ray induced block in deoxyribonucleic
acid synthesis. C. O. Doupney (introduced by
Stuart Mupp). Dept. of Microbiology, School of
Medicine, Univ. of Pennsylvania, Philadelphia.
Recent studies by Stapleton, Sbarra and Hol-

laender have demonstrated that a basal medium

supplemented with yeast extract or glutamic acid,
uracil and guanine will support an increase in the
number of survivors of Escherichia coli following
irradiation when compared to survival on non-
supplemented plates. Doudney and Hollaender
showed that an even greater level of survival oc-
curred following x-irradiation and plating on the
supplemented plates if the bacterial cells were
treated prior to irradiation with the chemical,
cysteamine (2-mercaptoethylamine). Further, it
was shown by Billen that an x-ray induced block
to DNA synthesis was overcome under conditions
of pretreatment with cysteamine and holding the
irradiated cells in yeast extract supplemented
basal medium. The present studies demonstrate
that the minimal supplement to the basal medium
in place of yeast extract supporting maximum
restoration from the x-ray induced block to DNA
synthesis of EZ. coli B/r include some 18 amino
acids and the purine, guanine and the pyrimidine,
uracil. The purine and pyrimidine may be deleted
with a 30% reduction in the rate of DNA synthesis

(net increase). Deletion of all amino acids except

tyrosine or phenylalanine, tryptophan, glutamic

acid or aspartic acid, and serine or glycine results
in only slight reduction of DNA synthesis. The
results suggest an involvement of protein synthesis
(in view of the amino acid requirements) and RNA
synthesis (in view of the requirement for guanine
and the RNA specific pyrimidine, uracil) in the
biosynthetic processes leading to restoration of
the x-ray induced lesion in DNA synthesis in E.
coli. (AEC Contract No. AT(30-1)-1342.)

163. Uptake of labeled glucose by tissue glyco-
gen in vivo. ARTHUR F. Dratz,* JANE A. Rus-
SELL AND BarBara M. Covey.* Radiosiotope
Service, VA Hosp., Atlanta, and Dept. of Bio-
chemistry, Emory Univ., Emory University,
Ga.

The uptake of relatively carrier-free C'*-glucose
by extractable and bound tissue glycogen in nor-
mal rats has been investigated. The rate of de-
crease of the specific activity of blood glucose in
fed rats was twice as fast as in fasted animals.
The glycogen specific activity time curves reached
maxima (3-25% of corresponding blood glucose
activities) in 1 to 3 hr. and then decreased. Gas-
trocnemius and cardiac glycogen fractions ex-
hibited appreciable uptakes in both fed and fasted
rats, the uptakes being considerably slower in
fasted animals. In these tissues the 2 fractions ex-
hibited substantially parallel activities, suggest-
ing a close metabolic relationship between them,
but some significant differences existed. In gas-
trocnemius muscle of both fed and fasted rats,
specific activities of extractable glycogen were
always somewhat less than those of bound glyco-
gen; but insulin treatment in fed rats, which in-
creased the activities of both fractions several fold
without alteration in analytical amounts, reversed
this relationship significantly. In cardiac glycogen
of fed rats, the specific activities were always
somewhat higher in extractable than in bound
fractions, and insulin produced no significant
changes. In fasted rats, the specific activity rela-
tionship of the fractions was reversed by increased
analytical amounts of extractable cardiac glyco-
gen. Liver glycogen exhibited virtually no uptake
in normal or insulin treated fed rats. In fasted rats
both liver glycogen fractions exhibited appreci-
able specific activities, that of the bound being
greater at early and less at later times.

164. Glucose utilization and lactate produc-
tion by leucocytes from diabetic and nor-
mal human subjects. Mary E. Dum. Dept.
of Medicine, College of Medicine, New York
Univ.-Bellevue Med. Center, New York City.
Carbohydrate metabolism was studied in leuco-

cytes from normal subjects and from patients with

diabetes mellitus. Unless otherwise indicated, the

diabetic subjects had not received insulin for 24

FEDERATION PROCEEDINGS

Volume jj

hr. before blood was drawn. Blood sugar valuegip
the diabetic subjects ranged from 150 to 342 mg%,
Aerobic glucose utilization at 37°C by leucocyte
in plasma averaged 9.5 mg (5-14)/hr/10° cells jp
6 normal subjects and 4.5 mg (1-9)/hr/10® eel
in 6 diabetic subjects. In general, the highest blog
sugar values were associated with the lowes
utilization of glucose by the cells. The addition of
insulin (0.1-0.5 u/ml) in vitro to leucocytes fron
normal subjects increased glucose utilization jp
7 out of 8 experiments. Insulin added in vitro t
cells from diabetic subjects increased glucog
utilization in 9 out of 11 experiments. The pr.
vious injection of insulin (2 diabetic, 1 normal)
increased the utilization of glucose by the leueo.
cytes and also resulted in a greater sensitivity of
the cells to insulin added in vitro. Lactate produe.
tion by the cells did not differ significantly between
the diabetic and normal subjects and was not af.
fected by in vitro insulin. The fact that defects in
carbohydrate metabolism occur in leucocytes from
diabetic patients indicates that the white blood
cells may serve as a source of living human tissue
in which to study metabolic lesions in these pa-
tients. (Supported by grants from the Josiah
Macy, Jr. Fndn. and the Natl. Vitamin Fndn.)

165. Lipid-metabolic alterations associated
with cortisone-induced regression of cho-

lesterol atherosclerosis in the _ rabbit,
ABRAHAM Dury. Dorn Lab. of Med. Research,
Bradford, Pa.

Aortic morphology, lipids’ distribution and re-
lationships and phospholipids’ metabolism in
aorta, plasma and liver were observed in normal
and cholesterol-fed rabbits. Cholesterol (crystal-
line) was mixed with shredded carrots and given
daily as a separate ration from the pellet diet.
This mixture was readily and entirely consumed
each day of the 4-mo. observation period prior to
killing the animals. Fourteen days before termina-
tion of the experiment, the rabbits on the regular
or cholesterol-supplemented diet were randomly
regrouped and assigned to receive daily intra-
muscular injection of 10 mg of cortisone or equiy-
alent amount of saline. This report is based upon
4 experimental groups: J, regular diet and period
of a) saline or b) cortisone injection; IJ, choles-
terol-supplemented diet and period of a) saline or
b) cortisone injection. P* was injected intraven-
ously in all rabbits 6 hr. before they were killed.
It was observed that rabbits of [7a had: 1) severe
and extensive aortic atherosclerosis; 2) total lipid
content was markedly elevated in plasma and liver
primarily due to greatly increased levels of es-
terified and free cholesterol and also increased
neutral fat, and to a much lesser extent increased
phospholipid in plasma but no change in liver
phospholipid content; 3) aortic esterified choles-

chole

event
this i
follov
neous
grouy
gle in

lume I

‘alues in
2 mg%,
1cocytes
cells in
10° cells
st blood
lowest
lition of
es from
ution in
vitro to
glucose
‘he pre.
normal)
e leuco-
ivity of
produe-
et ween
not af-
fects in
‘es from
e blood
n tissue
\ese pa-
Josiah
ndn.)

»ciated
»f cho-
rabbit.
esearch,

and re-
lism in
normal
crystal-
d given
et diet.
nsumed
prior to
ermina-
regular
ndomly
r intra-
r equiv-
2d upon
| period
choles-
aline or
traven-
. killed.
) severe
‘al lipid
nd liver
3 of es-
creased
creased
in liver
choles-

March 1956

terol particularly, free cholesterol, and phospho-
lipid content were greater than normal but aortic
neutral fat content was not altered; 4 turnover of
phospholipids of plasma, liver and aorta was sig-
nificantly increased compared with normal rab-
bits. Two weeks of cortisone treatment of cho-
lesterol-fed rabbits caused: 1) marked regression
or complete absence of aortic atherosclerosis; 2)
profoundly increased neutral fat content in plasma
and liver, and significantly decreased aortic neu-
tral fat content; 3) aortic and plasma esterified
cholesterol were significantly decreased, but
notably the plasma ester cholesterol still was 8
times greater than in the normal rabbit; 4) plasma
phospholipid amount and turnover rate were sig-
nificantly elevated but liver and aortic phospholi-
pid turnover rates were significantly less than rab-
bits fed cholesterol only; 5) plasma cholesterol to
phospholipid ratio was 3 times higher in group Ila
rabbits than in normals; but this ratio was de-
creased to 4 the value in the former group in
cholesterol-fed, cortisone-treated rabbits.

166. Effect of ascorbic acid in response of
blood glucose and adrenal cholesterol in the
rat to insulin. Murre, Dury* aNnD ABRAHAM
Dury. Dorn Lab. of Med. Research, Bradford, Pa.
The influence of exogenous high levels of as-

corbic acid on the response of the adrenal gland

and total organism to ‘stress’ has been variously
confirmed and denied. Presumably, high levels of
ascorbic acid interact with or modify biochemical
events which occur in tissues during ‘stress.’ In
this investigation changes in blood glucose were
followed at intervals up to 4 hr. after a subcuta-
neous injection of 0.24 u of insulin, regular, in
groups of rats previously administered daily, sin-
gle injections of 50 or 250 mg sodium ascorbate/100
gm b. wt. for 4 days. During the period of obtain-
ing tail blood, the rats were maintained under mild
anesthesia (combination of Nembutal and sodium
barbital) to avoid extraneous nonspecific ‘stress’
stimuli. At 2 and 4 hr. after insulin, groups of
tats were killed; heart blood, a specimen of liver
ind the adrenal glands were removed for deter-
minations of ascorbic acid, glycogen and choles-
trol, respectively. Preinsulin blood glucose levels
were not different in ascorbic pretreated and nor-
mal rats. The pattern of the postinsulin glucose
tirves and rates of decrement and recovery of
glucose concentrations were practically identical
in both types of animals. The maximum fall (ap-
prox. 50% of preinsulin level) in glucose concen-
tations occurred between 60 and 120 min. after
insulin. The glucose levels were not altered in as-
trbic pretreated and normal rats not injected
vith insulin and subjected to the same procedures

f obtaining tail blood. Liver glycogen, adrenal

wight and concentrations of adrenal cholesterol-

AMERICAN PHYSIOLOGICAL SOCIETY

total, esterified and free from ascorbic pretreated
and controls killed at 2 and 4 hr. after insulin were
not different. Blood ascorbic acid levels were 6-8
times greater in the ascorbic pretreated as com-
pared with normals.

167. Mixing curve of erythrocytes in the dog.
G. S. Eapre anp Ivan W. Brown, Jr.* Depis.
of Physiology and Pharmacology and of Surgery,
Duke Univ. School of Medicine, Durham, N. C.
Erythrocytes tagged with Fe®® were injected

into dogs, both intact and splenectomized, under

urethane-chloralose anesthesia. The subsequent
time-concentration curves show 2 simultaneous
phases of mixing, one periodic and short, the other

dropping exponentially and more slowly to a

constant level. These are thought to indicate 2

phases of capillary circulation, rapid and slow.

The slow phase cannot easily be explained by

elution of radioactive material, loss of cells dam-

aged in transfusion, cell destruction by anti-
bodies or sequestration of tagged cells in the
spleen. From data obtained it is possible to calcu-
late, on this basis, the volumes in the rapid circu-
lation (including arteries and veins) and in the
slow-circulation capillaries. Average values with
standard deviations obtained on 10 dogs were:
total circulating red cells 25.24.5.2 ml/kg b. wt.;
rapidly circulating red cells 21.8+4.7 ml/kg. The

slowly circulating cells were 4.72.4 ml/kg in 5

dogs that had not received a plasma expander

(oxypolygelatin) and 6.1+2.9 in 5 dogs given OPG,

but this difference was not statistically significant.

The rate of flow of cells into the slow circulation

was calculated to be 2.68+1.66 ml/kg/min. in 8

animals under chloralose-urethane anesthesia; 2

animals that did not receive chloralose averaged

24.8 ml/kg/min., which is significantly greater.

The rate of flow was not apparently affected by

OPG infusion.

168. Viscero-motor reflexes in the spinal ani-
mal. JoHN N. EBLE (introduced by I. M. Korr).
Dept. of Physiology and Pharmacology, Kirksville
College of Osteopathy and Surgery, Kirksville, Mo.
Reflex muscular responses to renal stimulation

have been studied as a contribution to the further

understanding of referred pain mechanisms and
autonomic-somatic interchange. The electromyo-
graphic responses of the longissimus dorsi to
visceral and various other stimuli in chordoto-
mized rabbits (C-8, T-1) are the subject of this
report. Catheters were placed in both ureters and
insulated wire electrode pairs were implanted at
various segmental levels of the longissimus dorsi.

Pinching a leg of this preparation resulted in uni-

form activity in all parts of the lumbar portion of

the muscle, concurrent with the stimulus, predomi-
nantly on the homolateral side. Stimulation of ex-

54 FEDERATION PROCEEDINGS

posed cutaneous branches of the posterior primary
rami resulted in reflex contraction of the longissi-
mus dorsi limited largely to the corresponding
segment. Gradual distension of a renal pelvis by
injection of water through its catheter also elicited
responses limited to specific segments. Thus, mild
distension produced a response on the homolateral
side in the region of L-2, L-3 or L-4 only. Further
distension increased the activity and caused a
spread to neighboring segments and occasionally to
the contralateral side. Such responses to renal
stimulation are characterized by a prolonged
after discharge. Reciprocal influences of somatic
and visceral stimulation on these reflex muscular
responses were also observed. (Supported in part
by grants from the Natl. Heart Inst. (H-1632) and
the American Osteopathic Assoc.)

169. Effect of exercise and coronary arterial
narrowing on growth of interarterial coro-
nary anastomoses. RicHARD W. ECKSTEIN.
Depts. of Medicine and Physiology, Western Re-
serve Univ. School of Medicine, Cleveland, Ohio.
Interarterial coronary anastomoses develop

after coronary arterial narrowing and occlusion

and in response to chronic hypoxia. Data as to the
role of exercise are not available. Circumflex ar-
teries of dogs were narrowed various degrees with
braided silk. One-half of the animals were kept at

rest in cages. The remaining dogs were exercised 1

hr. daily, 5 days weekly on a treadmill running at

4.7 miles/hr. at an incline of 30°. After 6 wk. the

left chest was opened and 2 cannulas were placed

in the circumflex artery dista] to the narrowed sec-
tion. The proximal cannula was directed centrally
and the distal cannula, peripherally. Blood flow
through the constricted segment (ligature flow)
was collected from the proximal cannula held at
the level of the heart, while arterial pressure was
maintained at the level existing after anesthesia
but befqre thoracotomy. Retrograde flow (col-
lateral flow) was measured from the distal cannula
held at the level of the heart, while mean arterial
pressure was adjusted to 100 mm Hg. India ink
was injected into the distal cannula after death
and the stained myocardium was isolated and
weighed. Ligature and retrograde flows were ex-
pressed/100 gm of myocardium/min. and plotted.

The data reveal that exercise substantially in-

creases collateral growth above that induced by

circumflex narrowing alone. (Work done during
tenure of Established Investigatorship of the

American Heart Assoc. and supported by the

Cleveland Area Heart Society.)

170. Colloidal behavior of high molecular
weight bacterial polyglutamates. H. EpEL-
HocH,* J. B. BaTeMAN AND C. B. THORNE.*
Dept. of Pathology and Oncology, Univ. of Kansas

Volume 1§

Med. School and Chem. Corps Biol. Labs., Camp

Detrick, Md.

The colloidal properties of several samples of
y-glutamyl polypeptide (GP) have been examined
by the methods of potentiometric titration, vig.
cosity, sedimentation velocity and light scatter.
ing. The samples differ with respect to their optical
isomer composition and their origin. A 100% p-
isomer was isolated from the capsule of B. anthra-
cis. Samples containing 50-50% and 90-10% of the
p- and L- forms respectively were obtained from
the medium of cultures of B. subtilis. The titration
data fit the modified Henderson-Hasselbalch
equation, ph = pk’ + n log [a/(1 — a)]. Both
pk’ and n decrease with increasing ionic strength.
In contrast to some non-peptide polyelectrolytes,
GP electrostatic effects are not eliminated by 0.1
mM NaCl. Viscosities have been measured as a
function of the degree of ionization (a). The vis-
cosity numbers, @;p/c, are convex towards the «
axis, indicating that the molecular domain of the
polypeptide is expanding rapidly when a is greater
than about 0.2. The slow expansion below this
value suggests that the contracted structure is
stabilized, presumably by hydrogen bonding. The
sedimentation data on anthrax GP in 0.10 m NaCl
show very strong concentration dependence when
the molecule is highly ionized and only slightly
smaller dependence in 1.0 m NaCl. Light scatter-
ing data confirm the conclusions drawn from vis-
cosity and sedimentation in showing that the
ionized forms of GP, even in the presence of large
amounts of neutral salt, are extended beyond the
root-mean-square distances of a random coil
configuration.

171. Gastrointestinal water and chloride. I.§.
EpELMAN, F. Gotcu,* N. J. Sweet anv J. Na-
DELL.* Univ. of California School of Medicine,
San Francisco.

The partition of body water and electrolytes
based on tracer dilution methods have emphasized
the need for precise definition of subdivisions of
the extracellular phase. Intraluminal gut water
and chloride were measured by direct analyses in
80 fasting rabbits. Total body-water and chloride
determinations were carried out using D.O and
Br®? as tracers. Ionic and molecular exchange of
chloride and water was evaluated from plasma:gut
specific activity ratios. Gut contents were col-
lected from the stomach, small intestine and prox-
imal half of the large bowel. Intraluminal gut
water averaged 121+3% of total body water, with
44+1%, 241% and 6+1.6% in the stomach, small
and large bowel, respectively. D.O exchange was
complete in the large bowel at 2 hr., in the small
bowel at 3 hr. and in the stomach at 4 hr. after in-
jection. Intraluminal gut chloride averaged 16+
4% of total body chloride, with 12+4%, 2.541%

a a ae ee ees a ey ee ee

on
Pan
hati

lume 1§
., Camp

ples of
‘amined
on, vis-
scatter-
optical
00% v-
anthra-
% of the
2d from
itration
selbalch
]. Both
rength.
rolytes,
1 by 0.1
das a
The vis-
ls the a
n of the
greater
ow this
cture is
ing. The
M NaC]
ce when
slightly
scatter-
rom vis-
hat the
of large
rond the
om coil

de. I.8.
> J. Na-
fedicine,

strolytes
phasized
isions of
it. water
alyses in
chloride
920 and
hange of
sma: gut
vere col-
nd prox-
inal gut
ter, with
sh, small
ange was
she small
after in-
ged 16+
2.541%

March 1956

and 1.5+0,5% in the stomach, small and large
bowel, respectively. Forty-one to 48 hr. after in-
jection, Br** exchange was 90%, 100% and 95%
complete in stomach, small and large intestine,
respectively. These data indicate that a significant
proportion of body water and chloride are in the
gut. This transcellular pool in the rabbit is equiv-
alent in magnitude to 50% of the combined plasma-
interstitial fluid volume and contains 30% as
much chloride.

172. Correlations between skin temperature
and blood flow in the foot of the dog. H. E.
EpERSTROM, T. VERGEER,* RussELL RHopE*
anD Paut Autness.* Dept. of Physiology and
Pharmacology, Univ. of North Dakota Med. School,
Grand Forks.

Blood flows were measured in the feet of anes-
thetized dogs by means of a cannula in the saphe-
nous vein. Simultaneous temperature readings
were taken on the footpad with thermocouples.
Data from more than 100 animals were analyzed in
an attempt to find the degree of correlation exist-
ing between venous outflow and temperature
changes. The average blood flow from the saphe-
nous vein in this group of animals was 11.9 gm/100
gm tissue/min. The average temperature readings
at this level of blood flow were as follows: footpad,
31.8; rectal, 37.6; air, 25.3°C. The circulation in-
dex, as calculated by Burton’s formula, was 1.1
for the average blood flow. When blood flow was
related to footpad temperature the correlation co-
efficient was +0.25, indicating only a moderate de-
gree of relationship between these factors. When
the temperature data were converted to the cir-
culation index a correlation coefficient of +0.53
between the index number and blood flow resulted,
indicating a closer relationship than with footpad
temperature alone. When blood flow was plotted
against footpad temperature it was found that a
tise of 1° was accompanied by an approximate
doubling of venous outflow. If blood flow was
plotted against the circulation index a rise of one
unit in the index was associated with an approxi-
mate quadrupling of venous outflow. These values
are not applicable when extremely high or low
levels of blood flow exist.

13. Growth changes used as assay method for
studying pancreatic functions. L. E. Ep-
WARDS AND A. C. BreHME.* Dept. of Physiology,
Med. College of Virginia, Richmond.

Pancreatic duct-ligated rats show a loss in
weight on a diet free of Pancreatin but again gain
wight on a diet containing 1% 3x Pancreatin.
As an assay method, these animals were placed
ona Pancreatin diet for 2 wk. and then on a no
Pancreatin diet for a similar period. The alter-
lating between Pancreatin and no Pancreatin

AMERICAN PHYSIOLOGICAL SOCIETY 55

was carried out for several months. This procedure
shows that some animals do not develop a com-
plete Pancreatin deficiency in the early weeks
after the operation. Although some animals, once
the deficiency is shown, will remain deficient,
others will recover from the deficiency even after
several months. A very small amount of pancreas
is necessary to remove a major portion of this de-
ficiency and, once initiated, regeneration is ex-
ceedingly rapid. This assay method is being used
to study fetal pancreatic function as well as to
study pancreatic extracts.

174. Intracellular distribution of DPNH
cytochrome c reductase in rat and guinea
pig spleen. Herbert J. Ercue (introduced by
L. Levensoox). Div. of Biological Chemistry,
Hahnemann Med. College, Philadelphia, Pa.

Rat and guinea pig spleen homogenates (0.25
M sucrose) were separated by differential centrifu-
gation into nuclear, mitochondrial, microsomal
and supernatant fractions. All fractions were as-
sayed spectrophotometrically for DPNH cyto-
chrome c reductase and cytochrome oxidase ac-
tivities and analyzed for N content. In the rat,
for homogenate, nuclei, mitochondria, micro-
somes, and supernatant fluid, reductase specific
activities (um cytochrome c reduced/min/mg N
at 25°) averaged 0.24, 0.13, 0.91, 0.79, and 0.07,
respectively, and the % recovery of activity in
the separated fractions was 26, 30, 40, and 7, re-
spectively. In the guinea pig, for homogenate,
nuclei, mitochondria, microsomes, and super-
natant fluid, reductase specific activities averaged
1.31, 0.73, 2.20, 5.19, and 0.19, respectively, and
the % recovery of activity in the separated frac-
tions was 21, 14, 55, and 5, respectively. Thus, the
reductase concentration (ratio of specific activity
fraction/specific activity homogenate) of rat
spleen mitochondria is considerably different from
that of rat liver mitochondria (HogEesoom, J.
Biol. Chem. 177: 847) while the reductase concen-
trations of guinea pig spleen mitochondria and
microsomes are similar to those of both rat liver
particulates. Isolation of rat spleen nuclei by the
sucrose-CaCl. layering technique reduced their
specific activity to 0.05 and their total activity to
only 5%, indicating that most of the nuclear ac-
tivity is due to mitochondrial contamination.
Cytochrome oxidase specific activities of rat and
guinea pig spleen mitochondria are about 6 times
greater than those of the original homogenates;
oxidase activities of the microsomes and nuclei
are considerably less than those of the homo-
genates. (Supported by AEC Contract No. AT (30-
1)-1069.)

175. Comparative effects of cortisone, hydro-
cortisone, and their dehydro derivatives,

prednisone and prednisolone, on anaphy-

lactic shock in mice. Jut1A M. EINBINDER,*

CuaRLES L. Fox, Jr. anp Cart T. NELSON.*

Dept. of Dermatology, Columbia Univ. College

of Physicians and Surgeons, and the Dept. of

Surgery, New York Med. College, Flower and

Fifth Avenue Hosps., New York City.

Studies were undertaken to assess the relative
efficacy of cortisone, hydrocortisone, prednisone
and prednisolone in the prevention of fatal ana-
phylactic shock in sensitized mice. Electrolyte
analyses of muscle, skin, viscera and bone were
also performed. These steroids in various doses
were administered intramuscularly at either 3,
6, 20, or 48 to 72 hr. previous to challenge with
antigenic material. On the basis of their compara-
tive action in preventing anaphylactic death, the
relative effectiveness of these compounds was de-
termined. From these results and from the tissue
analyses an attempt was made also to correlate
the chemical structure of these related steroids
with their physiological activity in this system.
a complete tabulation of these findings will be pre-
sented, including the data previously obtained
with ACTH, DCA and cortisone (Am. J. Physiol.
182: 518, 1955). (Aided by grants H-1650, Natl.
Insts. of Health, and DA-49-007-MD-628, Dept.
of the Army.)

176. Pattern of variation of intervals in the
discharge from muscle spindles and gamma
efferents. Earn Evprep, Curt Von EUvLer,
Tosuio KusaMA AND TOSHIHIKO TOKIZANE
(introduced by Ciara SzEeGo). Dept. of Anat-
omy, Univ. California at Los Angeles, and VA
Hospital, Long Beach.

Distribution curves have been plotted for the
duration of intervals in the single-unit discharge
from muscle spindles in the intact and deefferented
gastrocnemius of the cat and from single gamma
efferents, Curves of deefferented spindles closely
resemble chance distribution curves, although at
very low muscle tensions irregular patterns are
sometimes seen. At moderate and high tensions
the coefficient of variation of the intervals tends
to remain constant. Discharge from spindles with
intact motor innervation have markedly greater
coefficients of variation and commonly had skewed
or multimodal patterns. The coefficient of varia-
tion of those gamma efferents which are firing
spontaneously at constant mean rate is also large
and the curves approximate expected chance dis-
tribution. It is suggested that the irregularities
of the curves of afferents from spindles with in-
tact innervation may be the result of innervation
of individual intrafusal fibers by 2 separate mo-
toneurons.

FEDERATION PROCEEDINGS

Volume 16

177. Osmotic diuresis as a means to overcome
post-traumatic antidiuresis. Sv. Expryp.
JORGENSEN,* V. MrHasLov* AnD J. U. Scuuzggz,
Dept. of Surgery, Div. of Urology, Univ. of Roch.
ester, Rochester, N. Y.

The major cause for post-traumatic water re.
tention has been shown by several investigators to
be equal to the condition resulting from increaged
antidiuretic activity. In such cases the urinary
volume is determined by the ratio of solid intake
to water intake. Since the solid intake post-trau-
matically usually is infinitesimal, this would re-
sult in a diminished excretion of urine. A number
of patients during and following surgery received
intravenous fluids with varying amounts of solids
in the form of urea (2} to 5%). Approximately
3000 cc of i.v. fluids were given leading to a urinary
volume amounting on the average to 75% of the
intake. Less change in electrolytes and serum os-
molarity was seen than in surgical cases treated
with routine fluid administration. Also, weight
loss in the urea cases was the common finding as
opposed to little or no weight loss in the control
cases. The advantages of establishing an osmotic
diuresis post-traumatically rather than merely
restricting fluid intake appear as follows: 1) over-
hydration is difficult to achieve since excess of
fluid will be excreted because a sufficient amount
of solids is administered; 2) caloric intake need
not be restricted to the same extent as when fluid
intake has to be low; 3) increased excretion of
urine decreases the concentration of possible
nephrotoxic agents, thereby minimizing the pos-
sibility of renal damage; 4) in cases of lower
urinary tract infections, the danger of ascending
infections would be minimized with a high urinary
output. (Supported in part by contract NONR-
668 Task 7 from the Office of Naval Research.)

178. Comparative actions of a series of benz-
hydryl piperazines on mammalian hearts.
C. H. Exus, P. B. Russevi* anp L. N. Srvert-
sEN.* The Wellcome Research Labs., Tuckahoe,
Pe
Origin of auricular arrhythmias has _ been

ascribed to 2 mechanisms. Prinzmetal’s hypothe-

sis of multiple foci can account for the random
impulse formations of fibrillation, but Rosenbleuth
has clearly shown that circus movements do occur.

In view of the rhythmic nature of auricular flut-

ter, it does not seem unreasonable to assume that

flutter may result from re-entry of the impulse

giving true circus movements. If this be true, 4

compound which would lengthen the refractory

period of the auricular muscle without changing
conduction velocity should be effective in pre-
venting re-entry and thus should abolish the
flutter. A 3-stage screening procedure has been
used: 1) measurement of changes in refractory

earl

bloc

olume 15

ercome
ELDRUP-
HLEGEL,

of Roch-

ater re-
zators to
creased
urinary
d intake
»st-trau-
ould re-
number
received
of solids
cimately
urinary
Zo of the
TUM 08-
treated
- weight
nding as
- control
osmotic
merely
1) over-
xcess of
amount
ke need
ren fluid
etion of
possible
the pos-
of lower
scending
urinary
NONR-
rch.)

f benz-
hearts.
SIveRt-
'uckahoe,

s_been
:ypothe-
random
onbleuth
lo occur.
lar flut-
ime that
impulse
» true, a
fractory
hanging
in pre-
lish the
1as been
fractory

March 1956

period in isolated guinea-pig auricles, 2) assess-
ment of conduction velocity (changes in PR in-
terval in ECG in intact dogs) and 3) evaluation of
the ability of the compounds to abolish artificially
induced auricular arrhythmias in dogs. A series
of benzhydry] piperazines were investigated. Most
of these were much more active than quinidine.
The following substitutions seem to enhance the
activity which results in lengthening of the re-
fractory period: 1) alkylation of the ortho posi-
tion of one or both rings in the benzhydryl group,
9) methylation of the terminal nitrogen of the
piperazine ring. Among the compounds tested
omethyl benzhydryl methyl piperazine showed
no slowing of conduction as evidenced by the PR
interval, and was found to abolish experimental
auricular arrhythmias.

119. Acid-base alterations during hyperven-
tilation. James P. Exxis, Jr., J. GoRDOoN WELLS
anD Bruno BALKE (introduced by Husertus
StRUGHOLD). Dept. of Physiology-Biophysics,
USAF School of Aviation Medicine, Randolph
Air Force Base, Texas
During the course of performance studies di-

rected at determining the possible existence of

adaptation to hyperventilation, simultaneous
blood acid-base related measurements were made
on 6 male subjects. In addition to investigating
the basic physiological response to a standard
hyperventilation test, the influence of daily hyper-
ventilation training, physical conditioning and
altitude acclimatization was studied. Experi-
mentation on untrained individuals revealed that
a4-fold increase of ventilation could be endured
min., causing a 50% reduction in performance.
The blood pH increased from 7.41 to 7.57. Reduc-
tions of 15, 13 and 7% were found for plasma bi-
carbonate, plasma buffer capacity and the alkali
reserve, respectively. After 3 wk. of daily hyper-
ventilation training, improvement of endurance
ad performance was obvious. Similar changes in
blood were found for the first 30 min. as described
ibove. Continuation of the hyperventilation test
for the additional 30 min., however, caused the

pH to rise to 7.66, and reductions of 25, 15 and 10%

of plasma bicarbonate, buffer capacity and alkali

reserve, respectively. Eight weeks of physical
training did not alter the latter pattern of results.

Although absolute lactate, bicarbonate and buffer

values were reduced after acclimatization to 14,160

feet, relative changes during hyperventilation

fests were essentially reproduced. Tests made 2

ad 8 wk. after descent from altitude indicated a

gadual return of preacclimatization values. In

ill hyperventilation tests, blood lactate dropped

iightly during the initial 15 min. of hyper-

ventilation, but returned to, or exceeded, the

eer?

AMERICAN PHYSIOLOGICAL SOCIETY 57

resting value within 60 min. Blood pyruvate in-
creased throughout the test.

180. Excretion of epinephrine and norepi-
nephrine in various emotional states. FRED
ELMADJIAN, JusTiIN M. Hopre* anp Epwin T.
Lamson.* Worcester Fndn., Shrewsbury, and
Worcester State Hosp., Worcester, Mass.

Normal and psychotic subjects were studied
with respect to emotional state and excretion of
epinephrine (E) and norepinephrine (NE). After
acid hydrolysis the urine was extracted with the
alumina adsorption method of von Euler (Acta
physiol. scand. 22: 161, 1951) and bioassayed by a
modification of the method published by Gaddum
and Lembeck (Brit. J. Pharmacol. 4: 401, 1949). A
diurnal variation is observed in the excretion of
the catechol amines with higher values occurring
in the morning and the lowest during sleep. There
is a greater variation in the excretion of E than
NE with an average range of 1-4 meg/24 hr. for E
and 30-60 meg/24 hr. for NE. Using excretion data
obtained during infusion experiments, estimates
of the secretion of E and NE will be presented.
Studies were done on a) the players and coach dur-
ing professional hockey games, b) neuropsychiatric
patients appearing at staff conferences, c) normal
subjects in anticipatory states and d) psychotic
subjects receiving LSD. E and/or NE excretion
was observed to be increased under these condi-
tions. Data will also include urinary excretion of
catechol amines in psychotic subjects where psy-
chiatric rating scales were obtained during the
collection period. The results indicate that, in
general, the aggressive-hostile-active emotional
display is related to NE excretion, while the self-
effacing-fearful-passive display is related to E
excretion (Aided in part by a grant from the Army
Med. Research and Development Board, Contract
No. DA-49-007-MD-438.)

181. Effects of total profile and restricted area
exposure to 10-cm microwaves. THomas §.
Ezy anp Davin E. GotpMan (introduced by
Joun Z. Hearon). Naval Med. Research Inst.,
Bethesda, Md.

The increase in radar field power densities and
use of microwave therapeutic diathermy has gen-
erated interest in the hazard implications of this
form of energy. Most workers agree at this time
that biological effects are exclusively thermal.
The therapeutic diathermy and radar ‘S-Band’
frequencies, in the wavelength range of 10 cm, are
particularly hazardous because of localized heat-
ing. Longer wavelengths are absorbed more uni-
formly in tissue, with less tendency to produce
local areas of high temperature. Shorter wave-
lengths approach the infrared behavior, with
superficial absorption and consequent warning.

Investigations have been aimed at relating general
and local heating of experimental animals with the
microwave field intensity. Live mice, rats, rabbits
and dogs were exposed under relatively free field
conditions to 10-cm microwave energy from a
pulsed radar transmitter. Field intensity and dis-
tribution, time, local or rectal animal tempera-
ture, animal profile area and weight were measured
in a manner which permitted the estimation of an
absorption efficiency, steady-state’ heat dissipa-
tion ability at elevated temperature and cooling
time constant. Experience indicated that the body
as a whole, the eye and the testis were more sensi-
tive than any nonspecific restricted area and con-
sequently formed the limiting factors. Cooling
time constant and steady state heat dissipation
ability of each of the 3 areas enabled the formula-
tion of figures which relate time and intensity to
biological effect.

182. Autonomic responses to cerebellar
stimulation in the suprathalamic decere-
brate (decorticate) cat. J. D. Emerson,*
Joun M. Bruun, G. M. EmMerson* anv J. O.
Fotey.* Dept. of Physiology, Med. College of
Alabama and Univ. of Alabama School of Den-
tistry, Birmingham.

Data have been reported previously from this
laboratory which indicate that cerebellar stimu-
lation elicits autonomic responses in the lightly
etherized, curarized cat and in the intact unanes-
thetized cat stimulated through chronic elec-
trodes. To begin the delimitation of the neural
structures essential for cerebellar autonomic ac-
tivity, a total of 124 cerebellar points were stimu-
lated in 21 cats in which the cerebral structures
rostral, dorsal, lateral and lateroventral to the
thalamus had been removed by blunt dissection
under Pentothal anesthesia 1-2 hr. prior to stimu-
lation. Bipolar stainless steel electrodes 1 mm
apart in a LabTronies stereotaxic electrode holder
were used for stimulation. The biphasic stimulus
was delivered by a Grass S4A stimulator through
a SIU4A isolation unit. Most of the points were
stimulated 10 times at 3-5-min. intervals, with a
train of stimuli 4-20 sec. in duration at a frequency
of 200 eps. Facilitation of the micturition reflex,
inhibition of the micturition reflex, pupillary con-
striction, pupillary dilatation and retraction of the
nictitating membrane were obtained at specific
loci, as invariable responses (P = 10~ or less when
compared with interspersed nonstimulated con-
trol periods by group comparison or by chi? analy-
sis). (Supported in part by PHS Grant No. B-800
from the Natl. Inst. of Neurological Diseases and
Blindness. )

183. Antiketogenic effects of glucose and fruc-
tose in sodium fluoroacetate diabetes.

FEDERATION PROCEEDINGS

Volume ij

Frank L. ENGEL AND JOAN FREDERICKs!

Depts. of Medicine and Physiology, Duke Univ,

Durham, N. C. '

SFA, which blocks the Krebs cycle, indueg
temporary diabetes in the rat. The present study
was designed to determine whether the ketosis of
diabetes due to metabolic poisoning responds to
glucose and fructose as does that of pancreatic
diabetes. Twenty-four-hour fasted rats were poi.
soned with 0.6 mg SFA/100 gm i.p. and 4 or Ig
hr. later the antiketogenic response to saline,
10% glucose in saline and 10% fructose in saline
(1 ml/100 gm i.p.) was tested. Glucose and frue.
tose were equally antiketogenic in control rats,
Four hours aftr SFA, when ketone levels did not
yet exceed controls, saline and glucose had no
influence on ketonemia while fructose significantly
lowered ketone levels, but to a lesser degree than
in controls. Sixteen hours after SFA there was sig-
nificant ketosis. All treatments including saline
significantly depressed ketonemia but fructose
was somewhat more effective. Many of the ani-
mals were quite ill and difficult to bleed, suggest-
ing that they were in shock. This was confirmed by
finding low blood pressures and elevated blood
amino nitrogen levels. Since shock itself sup-
presses ketosis in rats the data were reanalyzed
by arbitrarily classifying animals as being in
shock if their blood amino nitrogen levels were
greater by 2 standard deviations than the mean
amino nitrogen of the corresponding unpoisoned
control. Rats in shock showed comparable de-
pressions in ketonemia with all treatments while
those not in shock by this criterion showed a
marked antiketogenic response only to fructose.
Thus SFA and pancreatic diabetes have compara-
ble antiketogenic responses to fructose and glu-
cose, respectively. Observations on the aati-
ketogenic effect of insulin in SFA diabetes are
indicated.

184. Effects of fasting and refeeding on com-
position of rat adipose tissue. Ceci Ev-
TENMAN, NELL S. AYRES* AND ALBERT R.
BEHNKE, JR. Biochemistry Branch, U. S. Naval
Radiological Defense Lab., San Francisco, Calif.
The amounts of fat, water and fat-free dry resi-

due in mesenteric, perirenal and genital] adipose

tissue were determined in adult Sprague-Dawley

female rats in 4 different dietary conditions: a)

without prior fasting, b) after a 4-day fast, c)

after a 4-day fast and 4 days of refeeding and d)

after 4 days of fast and 14 days of refeeding. The

diet used was Purina Laboratory Chow. A 4-day

fast resulted in a 20% loss in body weight and a

loss of 65-90% of the fat depots studied. Upon

refeeding, the rats regained the lost body weight
in 4 days, but the weight of the adipose tissues

did not reach prefasting levels in any instance

3:82 228-2 2

thu

“olume tj

RICKS,
ke Univ,

induces
nt study
etosis of
ponds to
increatie
vere poi-
| 4 or 16
> saline,
in saline
nd frue-
rol rats,
: did not
had no
ificantly
ree than
was sig-
ig saline
fructose
the ani-
suggest-
irmed by
1d blood
elf sup-
unalyzed
eing in
els were
he mean
poisoned
able de-
‘ts while
howed a
Fructose.
ompara-
and glu-
ne anti-
etes are

yn com-
cIL En-
ERT R.
S. Naval
», Calif.
dry resi-
adipose
-Dawley
tions: @)
fast, ¢)
g and d)
ing. The
A 4-day
it and a
d. Upon
y weight
e tissues
instance

March 1956

following 4.days of refeeding and only 3 of the
time in rats refed for 14 days. The changes in the
amounts of fat in the depots almost exactly
parallels the changes in the depot weights under
all conditions studied. Loss of fat accounted for
approximately 93% of the decrease in adipose
tissue. The relationship of the results obtained
to the alteration of body composition will be dis-
cussed.

185. Prevention of barbiturate withdrawal
convulsions in cats by cerebral electro-
stimulation. C. F. Essig anp A. WIKLER.*
Natl. Inst. of Mental Health, Addiction Research
Ctr., PHS Hospital, Lexington, Ky.
Observation and continuous activity cage re-

cording established the occurrence of 5-8 major

convulsions during the first 5-10 days after
abruptly withdrawing sodium barbital from each

of 3 cats addicted to 475-895 mg/day for 67-182

days. In 2 cats addicted to 760 and 855 mg for

30 and 16 days respectively, no withdrawal con-

visions occurred. Attempts to determine electro-

convulsive seizure thresholds during the absti-
nence period appeared to prevent spontaneous
barbital withdrawal] convulsions. Thus no with-
drawal seizures occurred in 4 cats which were
stimulated twice daily after addiction to 380-760
mg for 47-146 days. Transdural stimulation of the
hemispheres was delivered via implanted elec-
trodes with biphasic pulses of 2 msec. duration,
0/sec. frequency for 5 sec. the strength of which
was increased in 3-volt steps until convulsive
movements occurred. One cat which had 14 spon-
taneous withdrawal seizures was readdicted on the
same dose schedule to test the effect of electro-stim-
wation during abstinence. Electro-stimulation
3 times daily apparently prevented spontaneous
abstinence seizures during withdrawal. Although,
thus far, no electro-stimulated animal has had
spontaneous barbital withdrawal convulsions, ad-
ditional data relating dose and duration of ad-
diction to convulsive expectancy are necessary.

186. Some physical properties of the human
tibia. F. Gaynor Evans anp Mitton Lesow.*
Anatomy and Engineering Mechanics Depts.,
Wayne Univ., Detroit, Mich.

The ultimate tensile and shearing strength for
% and 79 specimens, respectively, of compact
bone of standardized size were determined. The
specimens from the anterior, posterior, medial
and lateral quadrants of the proximal, middle and
distal thirds of amputated unembalmed tibias of
Tadult white males, 23 to 61 years of age, were wet
when tested in a 5000-lb. capacity materials test-
ing machine calibrated to an accuracy of +1%.
The modulus of elasticity was also determined.
The distal third of the bone had the highest

AMERICAN PHYSIOLOGICAL SOCIETY 59

average tensile and shearing strength and modulus
of elasticity. The middle third was weakest in
tension while the proximal third was weakest in
shear and had the lowest modulus of elasticity.
The tensile strength and modulus of elasticity of
the distal third were 18% greater than that of the
middle and proximal third, respectively. Its
modulus was 6% greater than that of the proximal
third. The lateral quadrant had the highest aver-
age tensile and shearing strength and modulus of
elasticity. The anterior quadrant was weakest in
tension and shear and had the lowest modulus of
elasticity. The average tensile strength of the
lateral quadrant was 18% greater, its shearing
strength 21% greater and its modulus of elasticity
13% greater than the anterior quadrant. No con-
sistent relation between the physical properties
studied and the age of the individual was found.
(Supported in part by a research grant from the
Natl. Insts. of Health, PHS.)

187. Autoxidation of polyunsaturated fatty
acids in human plasma. JoHN D. Evans,
Nap1a L. OLEKSYSHYN AND JEROME M. WAL-
DRON. Physiology Dept., Temple Univ. School of
Medicine, Philadelphia, Pa.

A correlation has been made in human plasma
between autoxidation as determined mano-
metrically and tota] polyunsaturated fatty acids
as determined spectrophotometrically. From this
it is possible to formulate an empirical curve by
which one may estimate the total polyethenoid
acids in less than 1 ml of human plasma. The
procedure has the advantage that it is quick and
simple, but the limitation that it does not give
direct information about the individual poly-
unsaturated fatty acids.

188. Prolonged changes in lateral geniculate
potentials following optic nerve tetaniza-
tion. Epwarp V. Evarts,* JoHn R. Huaues*
AND WapeE H. Marsuna tt. Natl. Inst. of Mental
Health, Bethesda, Md.

Tetanic stimulation of the optic nerve in nem-
butalized cats produces two types of subnormality
of the postsynaptic lateral geniculate response:
1) an initial subnormality which lasts for several
seconds and 2) a prolonged second subnormality
which develops following recovery from the first
subnormality, and which may last for several
hours. The present report will describe certain
characteristics of this second subnormality (SS).
The optic nerve in nembutalized cats was stimu-
lated with single or tetanic (500/sec) near maxi-
mal pulses. Optic tract responses and geniculate
postsynaptic responses were recorded from the
lateral geniculate nucleus with a steel needle elec-
trode. Tetanization of the order of 0.5 min. pro-
duced an initial subnormality lasting several

60 FEDERATION PROCEEDINGS

seconds; this was followed by slight potentiation
of both presynaptic and postsynaptic responses
which lasted approximately 1 min. Following this
slight potentiation, SS developed and remained
for several hours. During SS the postsynaptic
spike amplitude was reduced to as little as 15%
of the pretetanic level, while the presynaptic
spike amplitude was not altered. Following teta-
nization for periods considerably less than 0.5
min., SS was, of course, less marked. Unex-
pectedly, however, SS was also less marked fol-
lowing very prolonged tetanization. Following
tetanization for 10 min. or longer, SS did not de-
velop. Moreover, when a tetanus of 10 min. or
longer was applied during SS produced by a pre-
vious shorter tetanus, the previously induced SS
was abolished and did not reappear during several
subsequent hours of observation. SS did not de-
velop in unanesthetized decerebrate preparations.

189. Time relationship between stimulation
of the hypophysis and release of ovulating
hormone in the rat. JoHN W. EVERETT.
Dept. of Anatomy, Duke Univ. School of Medicine,
Durham, N.C.

To test the possibility that neurogenic stimu-
lation of the hypophysis and release of ovulating
hormone (LH) may proceed concurrently in this
species (SAWYER AND Everett. Endocrinology
52: 83, 1953), hypophysectomies and atropine
blocking experiments were carried out in parallel
at progressively later times during the 2-4 p.m.
‘critical period’ on the day of proestrus. Para-
pharyngeal hypophysectomy was standardized
so that the gland was removed abruptly with
little bleeding 7-10 min. after introduction of
ether. None of the 35 hypophysectomized rats
retained any significant fragment of pars distalis,
as determined under the dissecting microscope
(X7) at time of autopsy. Three groups of 10-15
rats were hypophysectomized at approximately
2:45, 3:18 and 3:45 p.m. At corresponding times,
groups of 10-20 rats, totaling 51, were injected
subcutaneously with atropine sulfate (700 mg/
kg). The extent to which ovulation had been com-
pletely or partially blocked was noted at autopsy
on the morning after operation or injection. In
order of time, the percentages of animals thus
affected were: by hypophysectomy 90, 70 and 33;
and by atropine 55, 70 and 40. The time of LH
release thus closely approximates that of the
atropine-sensitive, presumably hypothalamic
mechanism. To demonstrate absolute concurrence
or a difference would require a more direct method.

190. Water and electrolyte balance in adrenal-
ectomized rats treated with cortisone and
hydrocortisone. W. J. Eversoie. Dept. of
Biology, Univ. of New Mexico, Albuquerque.
Male rats of the Long-Evans strain, weighing

approximately 200 gm, were adapted to a forced-

Volume i§

feeding regimen. The fluid diet was semisynthetig
and patterned after the medium carbohydrate
diet of Ingle (Am. J. Physiol. 147: 1946). Animals
were tube-fed 10 ml of diet twice daily and 12
ml of distilled water was injected into the stomach
between feedings. The rats were grouped and
treated as follows: group 1, adrenalectomized con-
trols; group 2, sham-operated controls; group $,
adrenalectomized and injected daily with 1 mg
cortisone; group 4, adrenalectomized and injected
daily with 1 mg hydrocortisone; group 6, adrena-
lectomized and injected daily with 2-4 ml of salt-
free adrenal cortex extract. Daily records were
kept for 5-12 days on urine volume and excretion
of sodium, chloride and potassium. Terminally
the animals were decapitated and serum obtained
for electrolyte determinations. Adrenalectomized
rats excreted more urine and sodium, but less
potassium, than intact ones. The blood serum of
adrenalectomized rats was low in sodium and high
in potassium. In adrenalectomized rats, neither
cortisone nor hydrocortisone reduced the urine
volume or corrected the sodium loss. Either hor-
mone caused a marked increase in potassium
excretion. Hydrocortisone caused serum elec-
trolytes to return toward normal more effectively
than did cortisone. Adrenal cortex extract fully
corrected water and electrolyte deficiencies in
adrenalectomized rats. These experiments indi-
cate that 11-oxy steroids are poor for maintenance
purposes in adrenalectomized rats because they
lack corrective effects in water and electrolyte
balance. However, different steroids affect elec-
trolyte balance in different ways.

191. Aldosterone secretion during bleeding.
Gorpon L. Farre.it, Rosert 8S. Rosnacigz
AND ELizABETH W. RavuscHKOLB (introduced
by J. M. WeRLE). Dept. of Physiology, Western
Reserve Univ., Cleveland, Ohio.

Male mongrel dogs anesthetized with sodium
pentobarbital were bled from the adrenal vein.
The adrenal venous blood was collected in sepa-
rate lots over consecutive 20 min.-periods for 3
hr. About 4 of the blood volume of each dog was
removed in this procedure. The lots of blood
representing corresponding time intervals from
each of 6 dogs were pooled for isolation of aldoster-
one and hydrocortisone by paper chromatographic
methods. In experiment I, the rates of aldosterone
secretion, expressed as yug/100 kg body wt/hr,
were 8.8, 10.8, 22.2, 26.7, 26.2, 15.2, 11.9, 22.5.
The rates of hydrocortisone were 17.5, 16.6, 25.2,
37.3, 28.3, 17.4, 20.6, 13.3, 12.6 ug/kg body wt/hr.
The ratio, maximum/initial secretion rate, of
aldosterone was 3.02 and of hydrocortisone was
2.23. In experiment IT aldosterone rates were 23.2,
22.5, 29.5, 20.6, 38.6, 40.4, 35.7, 29.4, 34.8. Hydro-
cortisone rates were 30.1, 30.0, 26.8, 21.6, 26.7,
33.4, 24.8, 24.4, 21.0. The ratio, maximum/initial,

(Ai

lume 1§

mthetie
hydrate
Animals
and 12
tomach
ed and
ed con-
roup 8,
h 1 mg
njected
adrena-
of salt-
Is were
cretion
minally
btained
;omized
ut less
rum of
nd high
neither
e urine
er hor-
fassium
n elec-
ctively
st fully
cies in
S indi-
penance
se they
trolyte

*t elec-

-eding.
SNAGLE
oduced
Western

sodium
1 vein.
n sepa-
s for 3
log was
~ blood
s from
doster-
zraphic
sterone
wt/hr,
), 22.5.
6, 25.2,
wt /hr.
ate, of
ne was
re 23.2,
Hydro-
5, 26.7,
‘initial,

March 1956

of aldosterone was 1.74; of hydrocortisone 1.12.
from this data it is concluded that some stimulus
js present in the experiment which initiates a
marked increase in aldosterone secretion. These
changes are closely related in time to alterations
in hydrocortisone secretion although the pro-
portionate increase in aldosterone secretion is
geater than that of hydrocortisone. Electrolyte
alterations do not appear to be the stimulus in
these experiments since no significant changes in
cither serum sodium or potassium concentrations
were found in similar experiments under the same
conditions. Preliminary experiments indicate
that the aldosterone response is prevented by the
infusion of large volumes of dextran in saline.

12. Effects of storage temperature on certain
physical-chemical properties of glycerol
pectate, a plasma expander. GrEorGE A.
Feicen, Ienatius L. Trapani* anp Mary S.
Hurp.* Dept. of Physiology, Stanford Univ.,
Stanford, Calif.

Glycerol pectate (Burke Research Co., ONR
Report No. 21, Jan. 1954; and Report No. 28,
Oct. 1954) is a polygalacturonic acid ester pro-
duced by treating a methanolic suspension of pec-
tic acid with glycidol, at 60rC. After subsequent
purification steps the material was lyophilized,
and received in that form by our laboratory. The
present experiments were made with Lot 16-A
which, according to the manufacturer, had an
esterification degree of 85%. Preliminary assess-
nent of the ‘unstored’ material included studies of
its titration behavior, and of the pH-dependence
of the viscosity and osmotic pressure. The ‘nat-
wal’ pH of aqueous glycerol pectate solutions was
55 to 5.8; 2.2. mEq NaOH was required to bring
1gm of glycerol pectate from px 5.5 to pH 7.4.
Titration on the alkaline side was difficult. Os-
motic pressure and viscosity were stable between
pH 3.0 and 5.3; above this limit there was a sharp
inrease in P/C and a somewhat more gradual
decline in (yn; — 1)/C. The number-average molec-
war weights and intrinsic viscosities of ‘unstored’
dycerol pectate and of samples stored in aqueous
wlution for 98 days at 2°, 23° or 37°C were esti-
mated by determining, for each case, the osmotic
pressures and viscosities over a range of glycerol
pectate concentrations. All measurements were
made at pH 4 using a Mcllvaine buffer system
f = 0.1 — 0.2, 37°C). The results presented be-
low were calculated by conventional formulas
from P/C and (yr — 1)/C values which had been
xtrapolated to infinite dilution.

Storage at n n
‘“Unstored” 61,619 0.430
2 30,951 0.305
23° 23 237 0.280
37° 22 ,082 0.230

(Aided by a contract between the Office of Naval
Research and Stanford Univ., NR 102060.)

AMERICAN PHYSIOLOGICAL SOCIETY 61

193. Synthesis of lipids from radioactive pro-
pionate by surviving adipose tissue slices.
D. D. Feuisr, E. Fetst* anp Rex L. Hurr.*
Radioisotope Service, VA Hosp., Univ. of Wash-
ington School of Medicine, Seattle.

Inguinal adipose tissue was obtained from male
Swiss mice fed an adequate diet ad libitum, sliced
and incubated in flasks designed for COz collec-
tion in the presence of the following C-14-labeled
acids (as sodium salts): formic, acetic, propionic
and succinic. Saponifiable lipides were recovered
by standard procedures and C-14 content deter-
mined in this as well as in the COz fraction.
Liver slices were used as a control tissue. The
results obtained indicate that surviving adipose
tissue is capable of synthesizing fatty acids from
propionate and does so to an extent 100-200 times
greater than does liver. In the case of adipose
tissue, propionate and acetate are converted to
fatty acids to a comparable extent, while in the
case of liver, propionate was converted to fatty
acids to a much lesser extent than was acetate.
In contrast, oxidation of propionate took place
to an extent comparable to oxidation of acetate
for both tissues. These results indicate that adi-
pose tissue might have a specialized metabolic
role in biosynthesis of fatty acids from propionate.
Neither formate nor succinate was converted to
fatty acids to any appreciable extent by adipose
tissue. These results exclude succinate as being
in the pathway of conversion of propionate to
fatty acids by adipose tissue. (Support by a grant
from the American Cancer Society.)

194, Inhibition of acetate metabolism by
short chain fatty acids in rat liver slices.
J. M. Feuts,* E. J. Masoro, Sytvra 8. Pana-
Gcos* AND Davin Rapport. Dept. of Physiology,
Tufts Univ. School of Medicine, Boston, Mass.
A previous report from this laboratory noted

the severe inhibitory effects of certain short

chain fatty acids on hepatic acetate metabolism.

Propionate at 0.004 M concentration almost com-

pletely suppressed the incorporation of acetate-

1-C* into CO: and fatty aci s. Butyrate at 0.004m

was effective in severely re ': cing the incorpora-

tion of acetate into CO, but h d a negligible effect
on C* incorporation into fatty acids. These in-
hibitions were thought not to .e the result of
isotope dilution. In an attempt to define more
clearly the nature of these inhibitions, the utiliza-
tion of acetate-1-C'4 has been determined. In
experiments with propionate the utilization of
acetate is severely depressed from control value of

65% to below 15%. Butyrate was also found to

inhibit acetate utilization from a control value of

70% to about 40%. In view of this degree of in-

hibition of acetate utilization, the unaltered in-

corporation of C4 into fatty acids would appear
to represent an actual increase in lipogenesis under

62 FEDERATION PROCEEDINGS

the influence of butyrate. Additional studies
have been conducted on the oxidation of C'*-
acetate, propionate and butyrate at various sub-
strate concentrations. The influence of acetate
and butyrate on C™ propionate oxidation has
been examined. Neither substance was found to
influence appreciably the amount of propionate
oxidized. In other experiments the effects of
acetate and propionate on C'‘-butyrate incor-
poration into CO, and fatty acids were studied.
Neither substrate was found to influence the oxi-
dation of C'* butyrate in liver tissue.

195. Effects of acute decompression stress
upon plasma and urinary potassium in
adrenalectomized dogs. FREDERICK P. FER-
GUSON AND Drietricn C. Smita. Dept. of Physi-
ology, Univ. of Maryland School of Medicine,
Baltimore.

Previous experiments of the authors (Am. J.
Physiol. 173: 503, 1953) have shown that exposure
of intact unanesthetized dogs to moderate hy-
poxia (30,000 ft. simulated altitude) for 90 min.
results in an average decrease of 19% in plasma K
concentration and an increase in urinary K ex-
cretion. Subsequent work has demonstrated that
exposure of these animals to severe hypoxia
(45,000-60,000 ft.) until the onset of respiratory
arrest consistently produces a marked rise in
plasma K concentration. In an attempt to deter-
mine whether the adrenal glands are essential
for these responses, the experiments have been
repeated upon bilaterally adrenalectomized dogs
maintained on cortisone or DCA. In 17 experi-
ments upon cortisone-maintained dogs, plasma
K concentration decreased by an average of 19.7%
during 90 min. exposure to 30,000 ft. In 16 experi-
ments upon DCA-maintained dogs, it decreased by
an average of 15.5% under the same conditions.
Urinary excretion of potassium increased during
hypoxia in both series of experiments. Exposure
of cortisone-maintained dogs to severe hypoxia
resulted in‘an increase in plasma K concentration
similar to that observed in intact animals under
comparable conditions. These results appear to
support the conclusion that, in dogs, the presence
of the adrenal glands is not essential for the
changes in plasma and urinary K observed during
acute decompression stress. (Supported in part
by the USAF under Contract No. AF 41(657)-21,
monitored by the USAF School of Aviation
Medicine, Kande!pn Field, Tex.)

196. Magnitude, variability and reliability of
regional sweating rates. I. D. FERGuson,*
A. B. HertzMan Anp A. J. RaMPonge.* Dept. of
Physiology, St. Louis Univ. School of Medicine,
St. Louis, Mo.

Sweating rates were determined on 10 regions in

6 healthy young adult males. Forty-two experi-

Volume 16

ments were carried out at chosen levels of cop.
stant ambient temperature (Ta), 6 being at each
of 90°, 100° and 115°F. The remaining 24 exp. were
at 110°F Ta. Determinations of regional sweating
rates were made for 2} hr. in each experiment,
The present results under rigorous climatic con-
trol confirmed and extended previous findings in
this laboratory (Federation Proc. 14: 71, 1955),
The 6 subjects showed differences in magnitudes
of regional sweating. However, no matter what
the temperature, the forehead was generally the
region of greatest sweating rate. Apart from the
forehead, sweating was most marked on the lower
extremity at 90°F Ta, but at both 110°F and 115°F
Ta sweating predominated on either the upper
extremity or the trunk. Variation (expressed as
coefficient of variation) in all regions was greatest
at Ta = 90°F. At any Ta, such variation was small-
est in the lower extremity. A single determination
was a reliable sample of the sweating rate on
any region for any one experiment no matter
what the Ta. In contrast, regions varied signifi-
cantly in their sweating rates on the same subject
on different days, but these repeat experiments
were only carried out at 110°F Ta. (Supported by
U. 8. Air Force Contract and Public Health
Service.)

197. Response in motility of noninnervated
smooth muscle to succinylcholine chloride.
JouN Ferauson. Dept. of Physiology and Phar-
macology, Creighton Univ. School of Medicine,
Omaha, Nebr.

A study was made of the effect of succinylcholine
chloride on motility of noninnervated smooth
muscle. Preparations from the amnion of the de-
veloping chick were made, usually one from each
specimen, and each suspended as a strip in 100
ml of warmed aerated Sollmann-Rademaeker’s
solution. Motility was recorded on a smoked
drum. This preparation contains muscle tissue
but is thought to be devoid of nervous elements of
any description. Most of the strips manifested
spontaneous rhythmicity. Some exhibited changes
in tonus. Different preparations showed some
variation in the degree of activity and reactivity
to drugs. Concentrations of anhydrous succinyl-
choline chloride in the bath ranging from 1:1,000,-
000 to 1:5000 were used. The spontaneously rhyth-
mic muscle responded to a concentration of 1:10,-
000 by increased rate of contraction and to 1:5000,
also by an augmented tonus. The drug, in any
concentration used, failed to depress spontaneous
activity in this muscle. Alterations in the osmotic
relations of the solution induced by the addition
of 100 mg of glucose did not modify significantly
spontaneous activity in fresh preparations. A
concentration of 1:10,000, or more, of succinyl-
choline chloride appeared to depress slightly
sensitivity to threshold doses of acetylcholine,

i a ae)

— ee a... |

s &@weaeoo

lume 1§

of con-
at each
Pp. were
veating
riment,
ic con-
ings in

1955).
nitudes
r what
lly the
om the
e lower
1 115°F

upper
ssed as
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ination
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rvated
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1 Phar-
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choline
smooth
the de-
m each
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ents of
ifested
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| some
ctivity
ecinyl-
:1,000,-
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f 1:10,-
1:5000,
in any
aneous
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Adition
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ons. A
ecinyl-
lightly
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March 1956

an excitant of motility in this muscle, but the
yariation in response of different preparations
precluded making quantitative determinations.
Moreover, after excitation by acetylcholine the
above concentration of succinylcholine induced a
slight depression.

198. Effects of mephentermine on cerebral
circulation and metabolism. R. W. Fereu-
son,* D. W. RicHarpson* anv J. L. PATTERSON,
Jr. Dept. of Medicine, Med. College of Virginia,
Richmond.

The common use of pressor amines for the treat-
ment of hypotensive states indicates a need for
studying their effects on the area most vulnerable
to reduction in blood flow, the central nervous
system. The synthetic pressor amine mephenter-
mine (Wyamine) differs from other pressor amines
in that it has no asymmetric carbon atom. The
effects of mephentermine on cerebral circulation
and metabolism were studied by the nitrous oxide
method in nine normal subjects. The mean values
for the cerebral functions before administration
of mephentermine were: cerebral blood flow (CBF)
58 cc/100 gm brain/min.; cerebral oxygen con-
sumption (CMRos) 3.4 cc/100 gm/min.; cerebral
vascular resistance 1.67 mm Hg/cc blood flow
/100 gm/min. Mephentermine was administered
by intravenous drip. The average total dosage
was 31 mg over a period of 26 min. The arterial
pressure was stabilized at a level 10 mm Hg
higher than control before the second cerebral
determinations were made. Mephentermine pro-
duced statistically insignificant changes in CBF
and CVR. In contrast, the CMRo: increased 22%
(P < .01). The cerebral arteriovenous oxygen
difference increased 8%. The principal untoward
symptom was a sensation of lightheadedness,
usually mild. It is concluded that mephentermine
has no cerebral vasoconstrictor action but is a
cerebral metabolic stimulant. A potential vaso-
constrictor action may have been offset by the
vasodilator effect of increased metabolism.
Mephentermine has a theoretical advantage over
nor-epinephrine, which produces moderate vaso-
constriction. These data should provide clues to
development of a pressor agent without cerebral
vasoconstrictor or cerebral stimulant effects.

199. Estimation of compliance of components
of thorax. BENJAMIN G. Ferris, JR. Dept. of
Physiology, Harvard School of Public Health,
Boston, Mass.

Measurements of compliance of lungs (Cz)
and thorax (Cr) have been made by many investi-
gators. The several components of the thorax
have not been so studied; therefore it was felt
of value to attempt to separate contributions of
different portions of the thorax. For the purposes
of this analysis components of the thorax were

AMERICAN PHYSIOLOGICAL SOCIETY 63

considered to consist of rib cage, diaphragm, and
abdominal wall and abdominal contents. Pres-
sures were sampled in esophagus and stomach;
with the subject in a tank respirator, pressure
differences could be measured across the dia-
phragm (esophagus to stomach), across the ab-
dominal wall and contents (stomach to tank),
and across the thoracic structures (esophagus to
tank). The volume exchange was measured at the
mouth with a spirometer. It was important that
the volume change be solely related to the pres-
sure difference across the structure being de-
formed. Because of this, measurements of dia-
phragmatic (Cp) and abdominal (Ca) compli-
ances were done with the chest tightly bound to
minimize rib-cage motion. Compliance of the
thorax (Cr) was measured with the rib cage un-
bound. The compliance of the rib cage (Cr) was
then obtained by calculation. For convenience
of calculation the various compliances were
treated as capacitances of a group of condensers
in an electrical circuit. In such a circuit the ab-
domen and diaphragm are in series and the rib
cage is in parallel with both. The compliance of the
rib cage can be obtained from the following for-
mula: Cg = Cr — CaCp/Ca+Cp. Values so ob-
tained on patients with respiratory muscle paraly-
sis due to poliomyelitis have shown that the
diaphragm is extremely compliant in the inspira-
tory direction to (Cp,a4 = 0.32 1/em H,0;
Conia = 9.09 1/em H.O). In adults the rib cage
is stiff (Ck = 0.015 1/em H.O), whereas in chil-
dren it is relatively more compliant (Cr = 0.054
1/em H;,O). As the diaphragm is forced into an
expiratory position there is a point in the dia-
phragmatic compliance where there is an abrupt
change and the diaphragm becomes uncompliant
(Co,auit = 9.016 1/em H20).

200. Respiratory movements of the vocal
cords. B. Raymonp Fink, Mitos BASEK AND
V. EpANcHIN (introduced by W. W. Watcort).
Columbia Univ., College of Physicians and Sur-
geons, New York City.

Rhythmic widening and narrowing of the glottis
during respiration is generally explained as due
to rotation of the arytenoid cartilage by the crico-
arytenoid muscles. X-rays and motion pictures
show no evidence of rotation during opening and
closing of the glottis. When opening, the entire
vocal process of the arytenoid moves laterally,
remaining parallel to its fellow except at the ex-
treme lateral end of its excursion. An appearance
of divergence then develops. This appearance is
the result of backward tilt of the arytenoid around
the oblique downward and outward axis of the
cricoarytenoid joint. At no time during closure
of the glottis is there any sign of internal rotation.
Simultaneous cinematography and electromyog-
raphy shows that contraction of the sterno-

64 FEDERATION PROCEEDINGS

thyroid and thyro-hyoid muscles accompanies
the rhythmic down-and-up excursions of the
larynx with respiration. The soft tissues of the
larynx are attached superiorly to the hyoid bone
and inferiorly to the cricoid cartilage and form
two folds, the vocal and ventricular folds, re-
sembling the arrangement in a bellows. X-ray
studies show that as the larynx moves down away
from the hyoid, the folds, or pleats of the bellows
are stretched and unfold, the arytenoids slide
laterally and the larynx opens. When the larynx
moves upward the pleats become folded, the
arytenoids slide medially and the lumen is oc-
cluded. It is concluded that extrinsic muscles
which cause the up-and-down movements of the
larynx play an important role in opening and
closure of the glottis during respiration. The
concept that respiratory glottic movements in
man are controlled, by the recurrent laryngeal
nerves appears to require modification.

201. A vascular factor in visual accommoda-
tion. Davin G. FLEemrne (introduced by O. O.
StoLanp). Dept. of Physiology, Univ. of Kansas,
Lawrence.

Rabbits were used to test the hypothesis that
the sympathetic nervous system may affect ac-
commodation by controlling the diameter of blood
vessels in the ciliary body, thereby altering the
size of the ciliary body, and in this manner in-
fluencing the tension exerted by the zonule on
the lens. An attempt was made to correlate
changes in blood flow through the ear following
superior cervical gangliectomy, with changes in
accommodation. Ear temperature was the index
for blood flow and skiascopy measured refractive
power. A substantial time correlation between
changes in ear temperature and refractive error
appeared. Preoperative ear temperatures were
identical. The lst postoperative day the ears on
the operated side averaged 2.5°C warmer than the
ears on the contralateral side. Within 3 days no
difference existed. The Ist postoperative day the
eyes on the side with the lesion were 1.25 diopters
less hyperopic than the eyes on the intact side.
Within 3 days the difference disappeared. One
month postoperatively the rabbits were given .5
mg Priscoline daily i.v. Prior to the Priscoline
test period, ear temperatures and refractive
errors were the same bilaterally. An average dif-
ference of 1.9°C between the ears and 0.82 diop-
ters between the eyes resulted, with the ears on
the operated side always the warmer and the eyes
always the less hyperopic. Six months later a
similar response was elicited from the same group
of animals by a second round of Priscoline injec-
tions. (Aided by PHS grant B-716(c).)

202. Glucagon and liver glycogen. Piero P.
FoA, Erminio Costa* anp Giorgio GALAN-

Volume 1§

sino.* Dept. of Physiology and Pharmacology,

Chicago Med. School, Chicago, Ill.

Glucagon is believed to cause hyperglycemia by
promoting glycogenolysis in the liver; however,
an actual decrease of liver glycogen has not been
demonstrated with certainty. Repeated injections
of small doses of glucagon appear to have no effect
on liver glycogen (GALANSINO et al.) while large
doses actually may cause an increase (Root),
Glucagon was injected intraperitoneally into nor-
mal, alloxan diabetic, adrenalectomized and hy-
pophysectomized rats. The results indicate that;
1) 2 and 6 hr. after the injection of glucagon there
is a marked decrease in liver glycogen which is
still noticeable 12 hr. thereafter; 2) 24 hr. after the
injection of glucagon there is a marked increase
in liver glycogen; 3) large doses of glucagon
cause a decrease in adrenal ascorbic acid; 4) in
alloxan diabetic rats glucagon causes the initial
decrease, but not the subsequent increase in liver
glycogen; 5) in adrenalectomized and in hypo-
physectomized rats in which liver glycogen is
already very low, glucagon has little or no effect;
6) in adrenalectomized and in hypophysectomized
rats treated with cortisone, glucagon causes the
initial glycogenolysis but the subsequent rise in
liver glycogen is small and probably not signifi-
cant. No direct effects of glucagon on muscle gly-
cogen were noted. It is believed that the primary
effect of glucagon is an acceleration of liver glyco-
genolysis and that the delayed increase in liver
glycogen is secondary to the secretion of insulin
and/or of the pituitary and adrenal cortical
hormones. Similar results were obtained with
epinephrine. (Aided by a grant from the Public
Health Service.)

203. Regulation of insulin secretion by carbo-
hydrates other than glucose. Piero P.
FoA, Grioreio GALANSINO,* Erminio Costa*
AND Gurpo Pozza. Dept. of Physiology and
Pharmacology, Chicago Med. School, Chicago, Ill.
It is known that an increase in blood glucose

concentration causes a release of insulin from the

pancreas, however, very little is known about the
effect of other sugars. Fructose was investigated
by means of 7 pancreatic-femoral cross-circulation
experiments. The donor dog was hepatectomized
to prevent the transformation of the injected
fructose into glucose. No evidence of insulin re-
lease was obtained following the intravenous
injection of fructose (1 gm/kg) into the donor
dog. This technique could not be used to study
the effect of galactose due to the lack of reliable
methods for the differential determination of

galactose and glucose in blood. For this reason 4

single 1 gm dose of galactose, dissolved in 20 ml

of water, was injected into the pancreatic artery

of 7 dogs. In this manner a high concentration of

lume 16

acology,

emia by
owever,
ot been
jectiong
10 effect
le large
(Root),
ito nor-
and hy-
te that:
on there
vhich ig
fter the
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lucagon
1; 4) in
> initial
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1 hypo-
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ses the
rise in
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r glyco-
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insulin
cortical
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- Public

carbo-
sRo P.
Costa*
gy and
igo, Ill.
glucose
‘om the
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ilin re-
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donor
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reliable
ion of
ason &
» 20 ml
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March 1956

the sugar was obtained in the blood reaching the
pancreas without causing a noticeable increase of
reducing substances in the blood of the general
drculation. In 7 experiments the injection of
galactose was followed by a decrease in systemic
blood sugar concentration averaging 31% (22-
9%). In 6 out of 7 experiments the maximum
elect was obtained 30 to 75 min. after the injec-
tion. In 1 experiment the galactose was injected
rapidly by mistake, resulting in an immediate
increase in blood reducing substances. This in-
crease probably masked and caused a delay in the
appearance of the hypoglycemic effect which was
noticed only after 150 min. The injection of 20
nl of saline was without effect. The results sug-
gest that galactose, but not fructose, stimulate
the release of insulin from the pancreas. (Aided
by a grant from the Public Health Service.)

#4. Color-shift response in turtle retina re-
lated to difference in wave length. ALEXAN-
pER ForBES, CHRISTINA ENROTH,* MARJORIE
NeyLanp,* Mary Susan GONGAWARE* AND
Heten W. Deane.* Biological Labs., Harvard
Univ., Cambridge, Mass.

It was previously reported that in the turtle
all-cone retina color shift evokes an electric re-
sponse, which cannot be abolished by balancing
intensities (J. Neurophysiol. 18: 517, 1955). Meas-
wing the potential of this response shows gain in
magnitude as the difference in wave length be-
tween the beams is increased. With a deep red
interference filter (peak transmission 675 my)
for the fixed color and a monochromator for the
variable color and with intensities adjusted to
equalize the on-effect b-waves evoked by the two
beams, the potentials of the shift responses (short
to long wave) appear to increase in a stepwise,
rather than continuous, fashion as the wave
length of the variable color decreases. The most
rapid gains in shift-response potential are found
4s the variable color passes through the approxi-
mate wave-length range 650-630 my (red-orange)
and the range 615-595 my (orange-yellow). The
teverse shifts (long to short wave) give smaller
responses, more difficult to evaluate, but they
appear to show a similar stepwise increase. The
stepwise increase suggests that at these wave
lengths the color shift produces a maximum in-
crease in the activation of new receptors.

25. Tissue protein-bound iedine in anterior
and posterior lobes of bovine pituitary
glands. Wiu.1aM C. Foster, J. F. McCLENDON
AnD L. O. Grimore.* Dept. of Physiology,
Hahnemann Med. College, Philadelphia, Pa.
Tissue PBI determinations by the method of
McClendon and Foster in pooled samples of whole
thicken, dog and cat pituitary glands revealed
values higher than the PBI levels in many other

AMERICAN PHYSIOLOGICAL SOCIETY

tissues. Because of the large size of the bovine
pituitary gland it was possible to determine the
tissue PBI in the anterior and posterior lobes of
separate glands. Pituitary glands were extirpated
from adult cattle, placed in acidulated water,
boiled for 1 min., homogenized in a Waring
blendor, and centrifuged. The PBI determination
was made upon the mince. The range of PBI in
the anterior lobe varied from 0.52 to 1.35 yg/gm
dry weight of tissue, with a mean of 0.81 ug. The
range of PBI in the posterior lobe varied from
0.87 to 8.33 wg/gm dry weight of tissue, with a
mean of 3.96 ug, showing the latter to contain over
four times the PBI of the former. The authors be-
lieve the tissue PBI to indicate the presence of
the thyroid hormone, as distinct from diiodotyro-
sine, and these data indicate that 83% of the total
pituitary PBI was present in the posterior lobe.
Since the posterior lobe is connected with the
anterior by nerve tracts, it seems possible that
PBI (thyroid hormone) in the posterior lobe may
suppress the formation of TSH in the anterior
lobe thru this medium. (Aided by a grant from
Natl. Heart Inst., PHS.)

206. Phosphaturic effect of parathyroid ex-
tract in the dog. JAMEs FouLxks (introduced
by Atrrep GitmMan). Dept. of Pharmacology,
Univ. of British Columbia, Vancouver.

The acute intravenous injection of 200 to 500 u
of parathyroid extract (PTE) (Lilly) does not
elicit phosphaturia in the unanesthetized fasted
dog. No increase in phosphate excretion is ob-
served 8 hr. after intramuscular injection of 300
u PTE. However, intramuscular priming doses of
300 u PTE permit the subsequent development of
considerable phosphaturia when additional PTE
is administered intravenously 8 hr. later. When
the additional PTE is infused at 0.6 vu/min.,
progressive phosphaturia develops during the
4th and 5th hr. of the infusion. When additional
PTE is provided by acute intravenous injection
of 200 u, the response is more immediate but sub-
sides within 3 hr. Acute intravenous injection of
200 u PTE is not effective in producing phospha-
turia when the priming dose given 8 hr. prior to
the period of observation is administered intra-
venously rather than intramuscularly. The phos-
phaturia produced by PTE is primarily the result
of altered renal tubular transport and is not de-
pendent upon an increased filtered load of phos-
phate. The data indicate that the phosphaturic
principle in PTE has an appreciable latent period
of action, and is inactivated fairly rapidly within
the circulation. The results suggest that main-
tenance of an adequate level of this principle
over a considerable period of time is more effec-
tive than the achievement of a transitory high
concentration in eliciting maximal phosphaturic

66 FEDERATION PROCEEDINGS

responses. The renal action of PTE does not occur
with sufficient rapidity to account for the imme-
diate adjustment in tubular phosphate transport
which underlies the phosphaturia following in-
travenous phosphate injection.

207. Comparison of arterial-dilution curves
of Evans blue and methylene blue. [RWIN
J. Fox,* Wriu1am P. Crow .ey, Jr.,* JosePu
B. Grace* anp Eart H. Woop. Mayo Fndn.
and Mayo Clinic, Rochester, Minn.

Dilution patterns were recorded by ear and
cuvette oximeters after successive injections
(average = 6 min. apart) of Evans blue (T-1824)
and methylene blue in 12 normal persons and 29
cardiac patients. In equal doses, methylene blue
produced peak deflections 1.9 (1.5-2.2) times
greater than T-1824 in normal persons. Concen-
trations of methylene blue in arterial blood 30
sec. after peak deflection decreased to 2.9 (1-4)%
of the peak as compared to 16.0 (12-20)% for T-
1824, while simultaneous concentrations at the
ears averaged 17 and 19% for methylene blue and
T-1824, respectively. These findings, along with
reduction or absence of systemic recirculation
peaks with methylene blue, demonstrated its
rapid loss from circulation. By means of ear
oximeters, methylene blue was demonstrated in
‘bloodless tissue’ (pressurized ears) 2.4 (1-4)
min. after intravenous injection (0.1-0.3 mg/kg
body wt.), while T-1824 was not so detected.
Arterial curves from 10 patients with left-to-
right shunts demonstrated decreased prolongation
of disappearance slopes with methylene blue as
compared to T-1824. Degrees of prolongation
of disappearance slopes expressed as ‘disappear-
ance slope ratio’ correlated well with the magni-
tude of left-to-right shunts. This ratio, having
peak concentration as denominator and concen-
tration at a point on the disappearance slope
lying a distance equal to the build-up time after
peak congentration as numerator, permits semi-
quantitative estimation of left-to-right shunts
from methylene blue or T-1824 curves. Methylene
blue was found useful in localization of right-to-
left shunts.

208. Treatment and prophylaxis of experi-
mental renal hypertension in monkeys with
renins and antirenins. M. H. Franx,* L.
GRAHAM* AND G. E. WaKERLIN. Dept. of Physi-
ology, Univ. of Illinois College of Medicine, Chi-
cago.

We have previously reported negative results
with high antirenin titers in the treatment and
prophylaxis of experimental renal hypertension
in monkeys with hog renin (Federation Proc. 13:
47, 1954). Since then six attempted modifications
of the antigenicity of semipurified hog renin by

Volume 1§

ormalin and other agents were found to be with.

out antihypertensive effect in monkeys and the
resulting antirenins did not neutralize human or
monkey renin. One renal hypertensive and 2
normotensive monkeys were treated with crude
human renin from cortex. A pressure decreage
well correlated with the antirenin titer (maxi-
mum 3.3 DAU/ml) occurred in the hypertensive
animal. One normotensive monkey had a titer
of 4.8 DAU at the time of unilateral renal artery
constriction and was protected while the other,
with 0.5 DAU, was not protected. In passive
transfer experiments with dog serum, large doses
of antirenin to hog renin were ineffective in 2 renal
hypertensive monkeys, although maximum titers
of 20 and 26 DAU were observed. Four monkeys
treated with small doses of antirenin to human
renin prepared in the dog exhibited decreases to
or toward normotension with maximum titers of
0.3-0.9 DAU. Antiserum nephritis did not de-
velop. Since antirenin to human renin neutralizes
both human and monkey renins, these results
support the hypothesis that renin or a closely
related protein is involved in the pathogenesis
of primate experimental renal hypertension,
Further studies are in progress. (Aided by the
Natl. Heart Inst., the American Heart Assn. and
the Chicago Heart Assn.)

209. Sympathetic innervation of the nose,
FLORENT E. FRANKE, PETER O. BRAMANTE*
AND Witma I. WinteER.* Dept. of Physiology,
St. Louis Univ. School of Medicine, St. Louis,
Mo.

The object was to determine the segmental
origin of the vasoconstrictor fibers from the thor-
acic spinal cord to the nose of the dog and to
evaluate their relative functional importance,
Changes in the caliber of the nasal blood vessels
were followed by recording comparable changes
in the volume of air within the closed nasal cav-
ities. The changes in nasal volume were recorded
with negligible pressure changes. The anterior
roots of the upper 4-6 thoracic spinal nerves were
exposed on one side, extradurally, by laminec-
tomy in 5 anesthetized dogs and were electrically
stimulated usually for 20 sec. (frequency 22.2/
sec.; duration of shock: 2.5 milliseconds; 9 volts).
Volume increases up to 2.0 cc were observed, after
latent periods of a few seconds. Stimulation of T;
and Tz gave the largest changes with a definite
tendency for a progressive decrease in responses
at lower thoracic levels. Stimulation of T; and
Ts may give or may fail to give prompt nasal
vasoconstrictor responses but sometimes gave &
delayed response. Intravenous injection of adren-
aline or stimulation of the splanchnic nerve re-
sulted in delayed vasoconstriction of the nasal
vessels. Stimulation of the cranial end of the

vay

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be with.
and the
aman or
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h crude
lecreage
' (maxi-
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a titer
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2 other,
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genesis
ension,
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nose,
LANTE*
stology,

Louis,

mental
e thor-
and to
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vessels
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anterior
'S were
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rically
2a
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, after
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efinite
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*; and
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March 1956

yagosympathetic or adrenaline intravenously
caused greater nasal vasoconstriction than the
stimulation of individual anterior roots. The
yolume changes observed were independent of the
level of the blood pressure.

210. Effect of variation of ambient tempera-
ture on spontaneous running activity of
normal and hypertensive rats. MELVIN J.
Frecty. Dept. of Physiology, Harvard Med.
School, Boston, Mass.

Ten male Sprague-Dawley rats of similar age
were used; 5 of these had a well developed hyper-
tension (180-200 mm Hg) resulting from bilateral
encapsulation of the kidneys with latex envelopes,
while 5 were normotensive (135 to 145 mm Hg).
These hypertensive rats ate well and could not be
distinguished by their appearance from the nor-
motensive rats. At 12 weeks after operation (22
weeks total age) all rats were transferred to stand-
ard wheel activity cages in a constant tempera-
ture room illuminated from 8 a.m. to 6 p.m. Am-
bient temperature was changed every 6th day.
The sequence of temperatures used was 25, 15,
30, 10 and 20°C. The mean spontaneous activity
of normal rats increased with decreasing tem-
perature in a sigmoid fashion ranging from 120
revolutions/24 hr. at 30°C to 980 at 10°C. Mean
spontaneous activity of hypertensive rats also
increased with decreasing ambient temperatures.
The mean activity ranged from 50 revolutions/
4 hr. at 30°C to 285 at 10°C. Though the activity
of hypertensive rats was less than that of normo-
tensives at all ambient temperatures used, differ-
ences in activity were statistically significant
only at 10, 15 and 20°C. These observations were
made with adult, inexperienced rats to exclude the
transitory increase in activity observed in younger
rats as well as to exclude differences in running
experience as a factor in the response to changes
of ambient temperature.

211. Vaseular and extravascular distribution
of radioiodoalbumin in mice. Jutius J.
FRIEDMAN (introduced by H. 8. Mayerson).
Biophysics Lab. and Dept. of Physiology, Tulane
Univ. School of Medicine, New Orleans, La.
Male albino mice of the CF-1 strain were in-

jected intravenously with radioiodoalbumin and

were killed by rapid immersion into liquid nitro-
gen at intervals of 5-750 min. post-injection.

When administered intravenously, iodinated al-

bumin disappears from circulation in a uniform

exponential manner for 1-13 hr., at which time the
tate of disappearance is greatly reduced. The
distribution pattern of radioiodoalbumin be-
tween the tissues is such that the tissues may be
divided into two groups. The first group includes
liver, kidney, spleen and lung; tissues which ex-

ern i

AMERICAN PHYSIOLOGICAL SOCIETY 67

hibit a rapid rise in radioactivity, reaching a
maximum within 5-15 min., after which the tissue
radioactivity proceeds to fall. The second group
of tissues includes intestine, skin and muscle and
is characterized by a progressive increase in tissue
radioactivity for about 1-1} hr., or until equilib-
rium is attained, after which time the concentra-
tion of tissue radioactivity declines. There is
considerable accumulation of radioactivity in
the extravascular spaces of the skin and muscle,
with less in the intestine, and little or none in
the liver, kidney, spleen and lung. Of the total
extravascular albumin, the muscle contains ap-
proximately 55%. This represents about 20% of
that contained in plasma or 12-15% of the total
exchangeable albumin. The extravascular albu-
min in the skin is approximately 40% of the total
exchangeable albumin, which represents about
12-15% of that contained in plasma or 8-10% of
the total exchangeable albumin.

212. Acid secretion by the perfused stomach.
M. H. F. FrrepMAN ANnpD H. Appert.* Dept. of
Physiology, Jefferson Med. College, Philadelphia,
Pa.

A method has been developed for studying in
dogs secretion by the stomach which is receiving
its éntire blood supply under controlled conditions
from an extracorporeal sources. All vascular
connections between stomach and body are in-
terrupted but the vagus supply is left inact. By
means of a pulsatile pump the stomach is per-
fused in situ with oxygenated blood collected from
the gastric portal vein or with fresh blood drawn
from a reservoir. Blood from donor dogs is used:
horse blood may be suitable also, but not human
blood. To obtain secretion of acid the tempera-
ture of the gastric blood and tissue must not fall
below 34°C. Perfusion pressure, measured at the
coeliac axis, must be above 100 mm Hg. A pulsa-
tile pressure (about 20 mm Hg) rather than a
constant pressure would appear preferable. To
maintain motor activities 3 hr. after commence-
ment of perfusion, the minimum volume flow
rate of blood must be above 100 ml/min.; to ob-
tain acid secretion the minimum rate is above 400
ml/min. Addition of histamine to the perfusion
system results in increased secretion of fluid which
usually is of constant chloride but of varying
hydrogen concentrations. Other factors regulating
secretion are presently under investigation.

213. Failure of absorption of esterified cho-
lesterol in absence of bile. Meyer FRIEpD-
MAN. Harold Brunn Inst., Mount Zion Hosp.,
San Francisco, Calif.

It has been postulated that bile acids aid cho-
lesterol absorption by promoting its esterification
either within the lumen or wall of the intestine.

68 FEDERATION PROCEEDINGS

To study this possibility, ‘physiologically esteri-
fied’ cholesterol in chylomicron form was obtained
by collecting the intestinal lymph of cholesterol
fed rats. Fifteen-ml quantities of this intact
lymph containing approximately 10 mg of esteri-
fied cholesterol then was introduced intraduo-
denally into rats with bile duct ligation and into
control normal rats, whose intestinal lymph then
was collected for 24 hr. Intestinal lymph also was
obtained from bile ligated rats given only normal
saline intraduodenally. The results of these ex-
periments indicated that even when cholesterol
was introduced intraduodenally in the same chem-
ical and physical state that it was found in intes-
tinal lymph, no significant absorption occurred
in the absence of bile. Thus the average cholesterol
content of the lymph collected from the bile duct
ligated rats given ‘physiologically esterified’
cholesterol was 6.2 mg, an amount essentially
the same as the 4.9 mg found in the control ligated
rats given only saline. In contrast, the lymph of
the rats with intact bile flow given ‘physiologi-
cally esterified’ cholesterol contained 15.4 mg of
cholesterol. Apparently, bile promotes the ab-
sorption of cholesterol by some means other than
mere esterification of the latter.

214. Influence of induced hypoxia on central
blood volume of normal man. H. W. Fritts,
Jr.,* E. BrRaunwaup,* A. P. FisHMAN AND A.
Cournanp. Dept. of Medicine, Columbia Univ.,
New York City.

The effect of acute induced hypoxia on the cen-
tral blood volume has been investigated by two
different methods. Method 1. T-1824 dye was in-
jected into the main pulmonary artery and a dilu-
tion curve inscribed by sampling arterial blood
through a recording densitometer. The volume of
blood between the points of injection and sampling
was calculated from the product of the mean cir-
culation time and the cardiac output. The output
was measyred both by the Hamilton dilution prin-
ciple and by the direct Fick technique. Although
the values of output agreed well (+10%) during
control periods, the agreement was less perfect
during hypoxia, the dye frequently giving a higher
value. In 7 of 13 subjects studied, the central
blood volume calculated on the basis of the dye
output increased from 100 to 400 cc during ar-
terial hypoxemia in which the oxygen saturation
varied between 85 and 68%. Method 2. The subject
was balanced on a tilt-table with the fulcrum at
the level of the diaphragm, and the relative
weights of the 2 ends of the body were compared
before and after inducing arterial hypoxemia
comparable to that used in method 1. Of 5 subjects
studied, none displayed an increase in the weight
of the head end of the body. The validity of the
2 methods will be discussed, together with the

Volume 15

possible effects of pulmonary blood volume and
flow changes on the variations in pulmonary ar.
tery pressure which are frequently observed.

215. Effects of dl-carnitine HCI on fatty acid
oxidation by liver homogenates from cho.
line deficient rats. Irvine B. Fritz (intro-
duced by Racumret LEvinE). Dept. of Metabolic
& Endocrine Research, Michael Reese Hosp.,
Chicago, Til.

Artom has demonstrated that livers from cho-
line deficient rats have a decreased rate of long-
chain fatty acid oxidation. While the in viv
administration of choline increased this rate, addi-
tion of choline in vitro had no effects (J. Biol,
Chem. 205: 101, 1953). In the course of the present
investigation this work has been confirmed. In a
continuation of studies reported recently (Acta
physiol. Scandinav., in press) on the effects of
muscle extracts on hepatic fatty acid oxidation,
it has been observed that the addition of dl-
carnitine HCl ((CH:)sNCH:CHOHCH.COOH)
in low concentrations (10-5 to 10-6 m) significantly
increased the rate of oxidation of C14-labeled pal-
mitic acid by liver homogenates in the presence
but not in the absence of ATP. Addition of carni-
tine resulted in a relatively greater increase of
label appearing in ketones than in COsz. The per-
centage increase elicited in the presence of carni-
tine was greater in livers from choline deficient
rats than in livers from normal rats. However,
the addition of carnitine did not elevate fatty
acid oxidation by livers from choline deficient
rats to the levels achieved in livers obtained from
deficient rats injected with choline 3 hr. before
killing. It therefore appears that the addition of
carnitine in vitro partially corrects the decreased
fatty acid oxidation system of livers from choline
deficient rats.

216. Respiratory acidosis after short periods
of oxygen breathing in emphysematous pa-
tients. HerMAN F. Froes,* CHar.es I. LErt-
wicu* AND Hurey L. Mottey. Cardio-Respira-
tory Lab., Univ. of Southern California School
of Medicine, Los Angeles.

A study was made to correlate the data on 3-
sec. timed vital capacity (TVC) and maximal
breathing capacity (MBC) with respiratory acido-
sis induced by oxygen breathing. The data were
analyzed on 206 patients having complete pul-
monary function evaluation. Measurements of
minute ventilation, arterial blood oxygen satura-
tion and pH were compared before and after
breathing 99.5% oxygen for 6-10 min. A fall in p#
below 7.38 was considered to indicate acidosis.
Respiratory acidosis on oxygen breathing o¢-
curred in only 2 instances where the MBC was
above 40% of predicted, but in 16 out of 75 pa-

SwBesrBSB Ee

—
S.
—_e

les

lume 15

me and
ary ar-
ed.

ty acid
n cho-
(intro-
etabolic
Hosp.,

m cho-
of long-
in vivo
e, addi-
’. Biol.
present
d. Ina
ry (Acta
ects of
dation,
of dl-
COOH)
ficantly
led pal-
resence
f carni-
ease of
he per-
f carni-
eficient
yweve;r,
e fatty
eficient
d from
before
tion of
creased
choline

veriods
us pa-
| Lert-
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School

2 on 3-
aximal
- acido-
‘a were
te pul-
nts of
satura-
1 after
] in pH
sidosis.
ng oc-
3C was
75 pa-

March 1956

tients with an MBC less than 40% of predicted
(av. 20% for the 16, group A, and 25.3% for the
other 59, group B). The mean arterial oxygen
saturation was 82.0%, pCOs 56.3 and pO: 62.0
mm Hg on air breathing in group A. The mean
minute ventilation was decreased on oxygen
breathing in group A from 3.62 to 3.34 1/min./
sq.m./BSA and the px from 7.41 to 7.34. In group
B the arterial saturation was 89.0%, pCO: 47 and
pO: 75.8 mm Hg, with no decrease in minute ven-
tilation on oxygen breathing and the pH change
was 7.45 to 7.42. The changes noted for TVC were
similar to those of MBC. The rapid development
of respiratory acidosis on oxygen breathing ap-
pears related to the pO: level and the decreased
minute ventilation. Emphysematous patients
may develop respiratory acidosis very quickly on
oxygen breathing if the TVC and MBC are less
than 40% of predicted, but otherwise rapid occur-
rence is infrequent.

217. Comparison of renal and hepatic effects
of fasting and cortisone in adrenalecto-
mized rats. E. Rupotr Froxscu, JAMES
ASHMORE AND ALBERT E. RENOLD (introduced
by Georce W. Tuorn). Depts. of Medicine and
Biological Chemistry, Harvard Med. School,
Boston, Mass.

Renal and hepatic carbohydrate metabolism
are qualitatively similar. In particular, both
tissues a) contain comparable amounts of glucose-
§-phosphatase, b) under certain conditions liber-
ate glucose into the blood and c) can deposit large
amounts of glycogen. It has been demonstrated
that hepatic glucose-6-phosphatase activity is
increased under conditions associated with in-
creased gluconeogenesis (J. Biol. Chem. 218:
17, 1955). This study reports a comparison of the
eflects of cortisone and of fasting on renal glyco-
gen and glucose-6-phosphatase activity with those
produced in the liver. Adrenalectomized male
tats were fasted 24 hr., then given 50 mg of corti-
sone over 24 hr. Liver glycogen increased markedly
from 0.3 to 54 mg/gm, whereas no deposition of
kidney glycogen occurred. Glucose-6-phosphatase
activity was measured in animals: a) maintained
on an adequate diet, b) fasted for 24 hr., c) given
cortisone (5-50 mg) during the 24-hr. fast, d)
treated with cortisone 24 hr. before fasting and
maintained on cortisone during the fasting period.
Fasting alone did not significantly increase glu-
cose-6-phosphatase activity of either liver or
kidney in these adrenalectomized animals. Ad-
ministration of cortisone for 48 hr. restored the
hepatic response to fasting to normal and a
parallel increase in renal glucose-6-phosphatase
activity was observed. That cortisone and fasting
lead to increased renal gluconeogenesis is sug-
gested by the similarity in hepatic and renal glu-

AMERICAN PHYSIOLOGICAL SOCIETY 69

cose-6-phosphatase changes. The 2 tissues differ,
however, with respect to their glycogen response.

218. Heat losses from human head in the
cold. Gerp Frorsre* anp ALAN C. Burton.
Lab. of Biophysics, Univ. of Western Ontario
Med. School, London, Canada.

Calculations suggest that there may be exces-
sive heat losses from the head in the cold. This
has been verified with a temperature gradient
calorimeter made of thin Styrofoam sheets. Re-
sistance measurements of wires covering the in-
side and outside surfaces of the walls gave the total
flux, which by Gauss’s theorem is proportional,
in the steady state, to the magnitude of the heat
source. The breath was led outside the calorim-
eter; thus heat loss from the head was measured
on 3 subjects (27 runs at 14 different room tem-
peratures). The average temperature over the
inside surfaces of the calorimeter walls ranged
from 32° to —21°C. Cheek temperatures were
measured in a few cases. A linear regression be-
tween heat loss and calorimeter temperature was
found, i.e. kg Cal/hr/m? of head surface (Du
Bois’ formula) = 295.5 — 7.90 to °C. The coeffi-
cient of correlation was 0.97 + 0.011. This would
indicate a heat loss at —11.3°C (9.4°F) equal to
half the total resting metabolic rate. Extrapolat-
ing to zero heat loss the ‘brain temperature’ was
found to be 37.4°C. This temperature was used to
calculate the insulation from the brain to the
calorimeter wall. An average value of 0.71 + 0.12
8.D. clo units was obtained. The contribution of
the tissue insulation to this total is estimated as
0.25 clo units. It appears, therefore, that there is
no detectable peripheral constriction in the head
during exposure to cold. This would seem to favor
keeping the brain temperature normal. (Sup-
ported by a grant from the Defence Research
Board of Canada.)

219. Electroencephalographic changes in the
cat associated with antidiuretic response to
intracarotid injection of hypertonic solu-
tions. GrorcEe R. Futter* anp Cuarues H.
Sawyer. Dept. of Anatomy, Univ. of California,
Los Angeles.

A study has been made of the effects on the elec-
trical activity in various cortical and subcortical
regions of the cat brain produced by injecting
hypertonic solutions into the common carotid
artery. Curarized female cats were employed,
and subcortical electrodes were placed stereo-
taxically. Hypertonic saline (0.5 m) and glucose
(2.4 mM) induced changes similar to those observed
by Sawyer and Gernandt (Anat. Rec. Feb., 1956)
in the rabbit—a 30-sec. triple response charac-
terized by initial arousal, intermediate depression
and final large amplitude slow waves. To trip this

70 FEDERATION PROCEEDINGS

mechanism in the cat, from 3-5 times as much
selution was required as in the rabbit, perhaps
because the cat lacks a functional internal caro-
tid artery. As in the rabbit, the most marked
EEG effects were observed in areas receiving ol-
factory projections. Hypertonic urea (2.4M), iso-
tonic saline and subeffective doses of hypertonic
saline served as controls. Intracarotid injections,
EEG observations and timed urine collections
were made during water diuresis, and we have
observed a good correlation between the ability
of a given injection to induce the EEG response
outlined above and its capacity to inhibit water
diuresis. Hypoxia and Nembutal both induce
marked antidiuresis as well as characteristic
EEG changes. Blood pressure changes are also
under observation. The results indicate that
‘osmoreceptor’ activation is associated with char-
acteristic electrical changes in areas far more
widespread than the supraoptic and paraventric-
ular nuclei—the neuroeffector sites for ADH
release.

220. Respiratory effects of Aminophylline
and Diamox alone and simultaneously in
pulmonary emphysema. Morton GALDSTON,
Jack GELLER,* VincENT Hows, JR.* AND
Eva LanpEesMan.* New York Univ. Research
Service, Goldwater Memorial Hosp., New York
City.

Studies are underway in moderate and advanced
emphysema patients of the effect of intravenous
0/5 G. Aminophylline (6.9-10.9 mg/kg), of Dia-
mox (3.3-10.4 mg/kg) every 12 hr., and of both
drugs together on gas exchange, arterial pCOz,
acid base balance, and on the sensitivity of the
respiratory regulatory mechanism to inhaled
CO:-O2 mixtures. Measurements were made
35-45 min. following Aminophylline, when respira-
tion was stabilized; and 1-3 hr. after Diamox.
In 5 patients the mean of 33 control arterial
pCO. measurements was 48 mm Hg. On Amino-
phylline it decreased 5 mm Hg (17 observations) ;
on Diamox 9 mm Hg (16 observations); and on
Aminophylline and Diamox administered simul-
taneously 13 mm Hg (13 observations). Mean
control arterial pH was 7.36. On Aminophylline
it remained unchanged or rose 0.04-0.06 units
(mean 7.40); on Diamox it decreased slightly or
as much as 0.07 units (mean 7.31). When Amino-
phylline and Diamox were given together Amino-
phylline prevented the diamox-induced arterial
pH fall in some patients (mean 7.34). On Amino-
phylline and on Diamox alone ventilation rose
in only 2 patients (11, 25%) and oxygen consump-
tion increased in only one (12%). When these
drugs were administered together ventilation
rose in all 5 patients from mean control 5.26 1/
min./M? to 7.67; however oxygen consumption

Volume 1§

increased in only two (15, 25%). R.Q. remained
unchanged. Diamox and Aminophylline each jp.
creased the ventilatory response to CO to about
the same extent; together, much more, without
altering the sensitivity of response.

221. Resistance of coenzyme A to radiation,
J. W. GarpELLA AND E. J. LIcHTLER (intro.
duced by J. C. Aus). Med. Labs., Collis P,
Huntington Memorial Hosp. of Harvard Univ,
Massachusetts General Hosp., Boston.
Knowledge of the in vitro radiosensitivity of

mercaptoenzymes and other sulfhydryl com-
pounds of biological interest has led us to investi-
gate the effect of radiation on the activity of coen-
zyme A. CoA activity of purified samples and
tissue extracts was measured by sulfanilamide
acetylation (Kaplan and Lipmann) and by the
phosphotransacetylase method (Stadtman et al.),
In vitro, pigeon liver CoA (courtesy Dr. Lipmann)
and commercial preparations (Pabst) in concen-
trations from 12.3-37.4 ug/ml and throughout a
pH range of 4.6-7.4 were not inactivated by as
much as 12,000r. The average content of CoA per
Yoshida ascites tumor cell was not altered signifi-
cantly by doses up to 10,000r delivered in vitro
to living cells. In vivo, 1000-1600r total body radia-
tion did not measurably alter, within 8 hr., the
pre-irradiation content of CoA of any of the fol-
lowing tissues (in units CoA per gram wet weight):
Wistar rat liver, 130; rat thymus, 18.8; Strain A
mouse liver, 112: Yoshida sarcoma, 7.6; duckling
liver, normal 81.4 and pantothenic acid-deficient,
45.5. Similar results were obtained when CoA
was assayed in a modified cysteine-free system
designed to detect disulfide and other oxidized
forms of CoA. Although CoA may be oxidized
or otherwise altered by irradiation as suggested
by Barron, these data indicate that its activity
as a cofactor in the acetylation of sulfanilamid
remains unimpaired.

222. Level of adrenal cortex extract necessary
to simulate lymphatic gland changes ocur-
ring in young chickens with fowl typhoid.
Henry W. Garren (introduced by F. N.
Craia). Poultry Science Dept., North Carolina
State College, Raleigh.

Fowl typhoid (Salmonella gallinarum) infection
in young chickens causes a marked increase in
the cortical activity of the adrenal which is ac-
companied by a marked involution of the thymus
and the bursa of Fabricius. Histologically, the
latter gland displays a loss of cells from the fol-
licles which seems to be closely correlated with
the degree of cortical activity. Studies were
conducted to ascertain the level of ACE (adrenal
cortex extract, Upjohn, 1 glycogen unit/ml) neces-
sary to cause the lymphatic gland changes ob-

~~ — . 2»

— |

inv

ean tad

olume 16

emained
each in-
0 about
without

liation,

(intro-
ollis P.
1 Univ.,

ivity of
1 com-
investi-
of coen-
les and
ilamide
by the
. et al.),
pmann)
concen-
zhout a
1 by as
SoA per
| signifi-
in vitro
y radia-
hr., the
the fol-
veight):
train A
luckling
ficient,
an CoA
system
»xidized
yxidized
ggested
activity
nilamid

pessary
$ ocur-
phoid.

FF.
Yarolina

fection
ease in
h is ac-
thymus
lly, the
the fol-
ed with
3 were
adrenal
) neces-
ges ob-

March 1956

served in chickens with fowl typhoid. For 6 days,
separate groups of Rhode Island Reds were given
daily injection of ACE in doses of }, 3, ?, 1, 2, and
3 ml. All levels of ACE resulted in a significant
decrease in the weight of the thymus and the
bursa of Fabricius. Two ml daily was sufficient to
cause both the decrease in lymphatic gland
weight and the histological changes in the bursa
of Fabricius equal to that noted for the group with
fowl typhoid. Most of the chickens given this
level of injection displayed a complete loss of
cells from the follicles of the bursa. Other changes
noted in the groups given 1, 2 and 3 ml of ACE
daily included: a decrease in body weight, an in-
crease in pituitary weight, a reduction in adrenal
weight (histologically, less cortical tissue), and
an increase in liver weight which became more
pronounced with the larger doses of ACE.

23. Effect of cooling and rewarming veloc-
ities in prevention of hemolysis by freezing.
P. M. Genenio* anp B. J. Luyet. Inst. of
Biophysics, St. Louis Univ., St. Louis, Mo.

It was reported previously that some 28% of
the erythrocytes were destroyed when small
quantities of blood (ox), spread in thin layers on
glass supports (microscope cover-slips), 0.14
mm thick, were frozen rapidly by immersion
in liquid nitrogen and rewarmed rapidly by im-
mersion in physiological saline at room tempera-
ture (LuyET, 1949). When the cooling and re-
warming velocities were increased by the use, as
supports, of small plates of perforated metal (an
aluminum alloy), 0.05 mm thick, the average
hemolysis was slightly less, namely, 23% (Luyetr
AnD GEHENIO, 1955). By increasing still further
both the cooling and the rewarming velocities,
we succeeded in reducing the hemolysis to about
}that value. We used as support a piece of alumi-
num foil 11 micra thick, spread, for the sake of
tigidity, over a thin-wire loop 25 mm in diameter.
When 0.05 ml of whole oxalated ox blood, spread
on this support, was frozen rapidly in liquid nitro-
gen (where it remained for 1-8 min.) and re-
warmed rapidly in physiological saline at room
temperature, some 14% of the red cells were de-
stroyed. By rewarming the biood in saline at 40°C,
instead of at room temperature, we could further
reduce the hemolysis to an average of 8%. (Sup-
ported by a research grant H-1826 (C) from the
Natl. Insts. of Health, PHS.)

%4,. Cytidine and uridine requirement ef
the brain. ALEXANDER GEIGER AND SEIICHIRO
YAMASAKI (introduced by R. GREENBERG).
Div. of Psychiatry, Neuro-Psychiatric Inst., Univ.
of Illinois, Chicago.

The nutritional requirements of the brain were
investigated in perfusion experiments on cats. It

AMERICAN PHYSIOLOGICAL SOCIETY 71

was shown previously, that the survival of the
perfused brain depends on substances released by
the liver into the blood circulation. In their ab-
sence aerobic glycolysis, followed by impermea-
bility to glucose developed, reducing the survival
time to about 1 hr. In recent experiments it was
possible to maintain normal brain functions in
the absence of liver for over 4 hr. by adding a few
milligrams of cytidine and uridine to the perfu-
sion blood. These substances were able to correct
the faulty carbohydrate metabolism of the brain,
which developed during an exhaustive perfusion
(with washed erythrocytes which were suspended
in Ringer-albumin) and restored the galactoside
and phospholipide content of the brain to the
level found at the start of perfusion. For main-
taining the physiological activity the presence of
both pyrimidine nucleosides was necessary. How-
ever, uridine alone restored the galactoside con-
tent of the brain to its original level after this has
been depleted by an exhaustive perfusion and
cytidine alone that of the phospholipides. It is
suggested that cytidine and uridine are essential
for maintaining brain functions, uridine acting
as a cofactor for galactoside metabolism, cytidine
for phospholipides. (Supported by PHS, grant
B.413.)

225. Effect of sodium barbital and metrazol
on adult brain cells in tissue culture. Rutu
S. Geiger (introduced by P. Battry). Div. of
Psychiatry, Neuro-Psychiatric Inst., Univ. of
Illinois, Chicago.

Addition of sodium barbital in concentration of
25 y/ml to the culture medium caused rapid intra-
cellular changes in the neurons and a slight con-
traction of the neuron. Practically the entire
cytoplasm appeared to become free of mitochon-
dria (phase contrast, vital staining with Janus
Green). The mitochondria, reduced in size and
more uniformly dispersed throughout the cyto-
plasm, began to reappear after 15 min. This
process was reversible on washing, the mitochon-
dria assumed their original size and distribution.
Recovery was accompanied by increased nucleolar
activity and concentration of perinuclear granules,
other than mitochondria. The granules in the glia
appeared unaffected by barbital. Addition of
Metrazol 200 7/ml. caused marked contraction of
the neurons and their nuclei appeared larger and
more transparent. The neurites were more active,
shortened, and became exaggeratedly beaded
with clumps of granules. During the first 10 min.
regions of differing refractivity became more
evident in the nucleolus, which exhibited an in-
creased motility. Many granules in the cytoplasm
aggregated into larger clumps and other accumu-
lated in greater concentration around the nuclear
membrane. Jacobson’s stain for nucleoprotein

showed these granules contained PNA; 15-20
min. after addition of Metrazol much of the ag-
gregated PNA-containing substance disappeared
from around the nucleus, fewer but larger clumps
were present in the rest of the cytoplasm, and the
nucleoplasm stained more weakly. Washing re-
sulted in a reversibility of these effects. In contrast
to barbital-treated neurons no changes were ob-
served in the mitochondria. (Supported by Illi-
nois D.P.W.)

226. Iodine-concentrating mechanism in skin
of the frog. JosrerpH F. GENNARO, JR. AND
Maraaret M. CLeMEnNTs (introduced by JAMES
B. Hamiuton). Dept. of Anatomy, State Univ.
of New York College of Medicine at New York
City, and Dept. of Zoology, Barnard College, and
Columbia Univ., New York City.

Disks of frog skin (Rana pipiens), 1 cm in diam-
eter, were incubated at room temperature in 5
ml of buffered Ringer’s solution to which 2 uc
of I's! had been added. The normal skin of dorsal
and ventral body surfaces was found to concen-
trate iodine and bind it in protein combination
(TCA precipitation). The effects of KI, TSH,
KSCN and thiourea on this system were deter-
mined. It was found that pretreatment of the
tissue with KI, thiourea or KSCN decreased the
radioiodide incorporation, especially in the pro-
tein-bound fraction. Incorporation of isotope was
enhanced in tissue incubated with TSH. Treat-
ment of the skin with KSCN was effective in dis-
charging incorporated radioactive iodine in the
white skin of the venter, but less so in the black,
heavily melanized dorsal skin, unless a tyrosinase
inhibitor (sodium diethyldithiocarbamate) was
added to the incubation medium. This would sug-
gest an association between the iodine-concentrat-
ing mechanism and those involved in the bio-
synthesis of melanin. It is concluded that the
iodine-concentrating mechanism in the skin of
the frog closely resembles that of the vertebrate
thyroid gland.

227. Sensitivity of paramecium caudatum to
oxygen toxicity as influenced by cobaltous
and manganous ions and hematoporphyrin.
R. Gerscuman, D. L. Gripert,* 8. W. Nyz*
AND W. O. Fenn. Dept. of Physiology, Univ. of
Rochester School of Medicine and Dentistry,
Rochester, N. Y.

Arrangements were made for the microscopic
observation of paramecia while they were exposed
to high oxygen pressure. This biological system
has the advantage of avoiding circulatory and
C.N.S. factors which may be involved in experi-
ments with mammals. Survival times were meas-
ured at different pressures of oxygen and tests

FEDERATION PROCEEDINGS

Volume 1§

were made of the effects of various substances re.
ported to be protective or detrimental in x-irradj-
ation or oxygen poisoning. The purpose of this
was to obtain further information on possible
similarities between x-irradiation and oxygen
poisoning (GERSCHMAN et al. Science 119: 62%,
1954). Opposite effects of the same agent often oe-
curred at different oxygen pressures. Thus, eo-
baltous chloride (M/20000) was protective at
5-20 but injurious at 14 atm. O2. Manganoug
chioride (M/2000) was protective at 9 but ap-
peared to be detrimental at 2 atm. Oc. Hemato-
porphyrin (1:200000) (which increased the sensi-
tivity to x-irradiation—F. H. J. FieGe an
R. WicHTERMAN. Science 122: 468, 1955) was
detrimental at 9 atm. Previously, we have found
cobalt protective to mice at 1 atm. O2 but detri-
mental at higher pressures. These experiments
emphasize the importance of studying the effects
of a given agent at different pressures of oxygen as
well as at different intensities of radiation.

228. Metabolic and tropic effects of human
pituitary glands. HERBERT GERSHBERG, Mal-
comm §8. Druskin* anp Mitton AGULNEK,*
Depts. of Medicine and Physiology, New York
Univ. College of Medicine, New York City.
Many diverse metabolic effects of growth hor-

mone have been described on the basis of the re-

sponse of rats and dogs to purified beef and hog
pituitary extracts. To our knowledge there are no
reports on the metabolic effects in these animals
of human pituitary glands. In view of this, saline
suspensions of frozen human pituitaries from 4
males, 46, 57, 57 and 60 yr. of age, who suffered
sudden, accidental deaths, were injected into
immature hypophysectomized rats. Our results
indicate that tropic activity is maintained in
glands kept frozen for long periods (6-7 mo.). The
body, heart and kidney weights and the width of
the tibial epiphyseal cartilage increased rapidly,
plasma inorganic phosphate increased and skeletal
muscle and cardiac glycogen were maintained on
fasting. The weight of the testes increased mark-
edly. The adrenal weight also increased and the
adrenal cholesterol concentration decreased, in-
dicating adrenocorticotropic activity. These ef-
fects could be obtained, in varying degrees, with
injection of as little as 2 mg pituitary tissue (wet
wt.) per day for 5 days. It is concluded that sub-
stantially large amounts of growth hormone occur
in pituitaries of older adults. (Aided by a grant
from the Air Force, Contract No. AF 41(657)-19).

229. Effect of head x-irradiation in rabbits on

aortic blood pressure, brain water content #}

Dr Nein a) net

aft

and cerebral histology. Hersert B. GERst- ep

NER, Paituips M. Brooxs anp F. STEPHEN

olume 16

ances re-
X-irradi-
2 of this
possible

oxygen
119: 623,
often oe-
‘hus, co-
ctive at
inganous
but ap-
Hemato-
he sensi-
iGE AND
55) was
ve found
ut detri-
eriments
1e effects
XY gen ag
n.

human
RG, Mat-
}ULNEK,*
‘ew York
ty.
wth hor-
yf the re-
and hog
re are no
, animals
is, saline
s from 4
_ suffered
ted into
r results
ained in
no.). The
width of
rapidly,
1 skeletal
ained on
ed mark-
and the
ased, in-
[hese ef-
ees, with
ssue (wet
that sub-
ne occur
r a grant
(657)-19).

bbits on
content
. GERST-
STEPHEN

March 1956

Voce. (introduced by R. W. BaAncrort).

Dept. of Radiobiology, USAF School of Aviation

Medicine, Randolph Air Force Base, Texas.

Rabbits received to the head 15 kr x-radiation
st a dose rate of 0.25 kr/min. The animals were
observed during irradiation and for 3 hr. follow-
ing the end of exposure when they were killed and
the brain excised for further processing. During
the 3-hr. postirradiation period, severe epileptoid
gigures appeared in nonanesthetized animals,
yhereas they did not occur in rabbits under ure-
thane anesthesia. Continuous blood pressure re-
wordings revealed a marked hypotension develop-
ing about 30 min. after start of irradiation. The
mean specific gravity of the irradiated brains was
10845 as compared to 1.0363 found for nonirradi-
sted controls; the water content determined with
sfreeze-dry method was 80.4% for the irradiated
rains and 78.4% for the controls; both differences
vere highly significant statistically. Histologic
studies showed early stages of developing menin-
itis, vasculitis and pyknosis of the granule cells
ifthe cerebellum.

0. Relationship between pH, chloride and
inulin in proximal tubule fluid of necturus
kidney. GERHARD GreBIscH (introduced by
R. C. Swan). Dept. of Physiological Chemistry,
Woman’s Med. College of Pennsylvania, Phila-
delphia.

Micropuncture studies such as those conducted
by Walker et al. (Am. J. Physiol. 118: 121, 1937)
lave indicated that the chloride concentration of
proximal tubule fluid is almost always higher than
that of serum. With no increase in tubular osmotic
jressure these results imply possibly preferential
rabsorption of bicarbonate and, assuming equal
O02 in serum and tubule fluid, some proximal
widification. To investigate the relationship be-
ween chloride concentration and acidification,
imultaneous measurements of chloride, inulin
ind pH were carried out on samples of fluid ob-
fined by micropuncture from proximal tubules
i Necturi. The concentration of inulin in tubule
fuid always exceeded that of serum, indicating
absorption of fluid from this part of the nephron.
The results obtained indicate that the proximal
tubule does not partcipiate in urinary acidifica-
ion and confirm the findings of Montgomery et
. (Am. J. Physiol. 118: 144, 1937). The concen-
tation of chloride in proximal tubule fluid when
tated with deproteinizing agents was found to
snot significantly higher than that of the serum.
thus no evidence of preferential reabsorption of
itarbonate could be demonstrated in this species.
lhe results presented resolve some of the dis-
tepancies of earlier micropuncture studies and
te in good agreement with known data on osmotic

AMERICAN PHYSIOLOGICAL SOCIETY

pressure and sodium concentrations in proximal
tubule fluid of Necturus.

231. Effects of high oxygen pressure (HOP) in
in vitro systems. D. L. GitBert,* R. Gerscu-
MAN AND W. O. Fenn. Dept. of Physiology,
Univ. of Rochester School of Medicine and Den-
tistry, Rochester, N. Y.

We have previously reported that exposure of
desoxyribonucleic acid (DNA) in the presence of
reduced glutathione (GSH) to HOP for about 12
hr. resulted in a viscosity decrease with a net
formation of H:O2. Further work has shown that
when 3.26 mm GSH/1. was exposed to 6 atm. O2
for about 14 hr. only 3 was oxidized, but at 130
atm. O2 no reduced glutathione was detectable.
It was of interest to investigate the effects of
adding various agents, which have been used in
in vivo oxygen toxicity studies, to these in vitro
systems. Thiourea inhibited the net formation of
H.0:2 in GSH solutions exposed to HOP and also
restrained the fall in viscosity produced by HOP
in the DNA solution containing GSH. Limperos
and Mosher found that thiourea also inhibited the
viscosity decrease of DNA resulting from x-ir-
radiation. In mice, thiourea has been reported to
protect against HOP and x-irradiation. Ethylene-
diaminetetraacetic acid disodium salt (EDTA),
a known chelating agent, also prevented the fall
in viscosity of solutions containing DNA and GSH
when exposed to HOP. EDTA at a concentration
of 0.3 um/l. prevented oxidation of GSH exposed
to HOP which would strongly indicate that its
action is due to removal of trace metals. Yet to
decrease oxidation of GSH, 3 m/l. of diethyldi-
thiocarbamic acid sodium salt, another chelating
agent, were necessary. The suggestion that some
chelating agents may be free radical receptors
should be given consideration in attempting to
interpret these results.

232. Effectiveness of various infusions in re-
storing cardiac output of the dog after
thermal injury. J. P. GrumMore (introduced by
StantEy W. Hanprorp). Dept. of Physiology,
Naval Med. Field Research Lab., Camp Lejeune,
N.C.

In a previous paper (Federation Proc. 14: 57,
1955) it was reported that, in the case of the dog,
the concept of loss of plasma volume causing a
decrease of cardiac output in severe burns appears
no longer tenable. It was shown that, in burn
shock, the decrease in cardiac output is not pro-
portional to the reduction of plasma volume; in
fact, a precipitous drop in flow may occur with
relatively little change of plasma volume. In view
of this it appeared a propos to evaluate the ef-
fectiveness of various infusions in restoring car-

74 FEDERATION PROCEEDINGS

diac output and plasma volume after a severe burn
in the dog, to determine whether increases of
volume would effect parallel increases of flow.
The results of these experiments demonstrated
that good restoration of plasma volume does not
necessarily mean good restoration of cardiac out-
put, nor conversely, does restoration of cardiac
output necessarily indicate restoration of plasma
volume. Of the infusions evaluated, plasma and
dextran appeared to be equally effective in re-
storing cardiac output and plasma volume in the
burned dog, dextrose-saline least effective and
gelatin intermediate.

233. Influence of temperature, anaerobio-
sis, iodoacetate and cyanide on phosphate
release from dog erythrocyte. JoHn M.
Ginsk1,* JoHN F. THomMson* AND AKIRA
Omacui. Argonne Natl. Lab., Lemont, and Dept.
of Physiology, Loyola Univ., Chicago, Ill.

In order to gain some insight into the mecha-
nisms governing the release of phosphate from
mammalian cells, the release of P*? from erythro-
cytes previously labeled with radiophosphate was
investigated. P*2 was incorporated into the cor-
puscles by incubating blood from unanesthetized
dogs for 2 hr. in the presence of added heparin,
glucose, phosphate buffer and radiophosphate.
The radiophosphate was obtained by eluting the
orthophosphate zone following electrochromato-
graphic separation. The erythrocytes were col-
lected by centrifugation in the cold, washed twice
and resuspended in fresh plasma. Aliquots were
transferred into Warburg flasks which were gassed
with O2:CO2 or N2:CO:2 (95:5) and P*? release
from the radioactive erythrocytes was determined
by analyzing plasma samples at frequent intervals.
Measurements of phosphate concentration,
hemolysis, pH and hematocrit were also performed.
Under both aerobic and anaerobic conditions, the
rate of phosphate release increased with tempera-
ture and activation energies (Arrhenius) of 2 x 104
cal/mole have been calculated. However, the
anaerobic rate was considerably lower, e.g. at
37°C phosphate was released at } of the aerobic
rate. Under aerobic conditions, iodoacetate in-
creased the rate of release, whereas cyanide had
no effect. These findings indicate that the rate of
phosphate release is governed oy metabolic proc-
esses; moreover, reactions requiring oxygen
appear to be involved as well as those concerned
with anaerobic glycolysis.

234. Excretion of sodium-retaining factor by
cortisone-treated, adrenalectomized hy-
pertensive patients. R. J. GrrERp* anp D. M.
GREEN. Univ. of Southern California, Los
Angeles.

Seven measurements of the 24-hr. urinary ex-

Volume i§

cretion of sodium-retaining factor were made in3
totally adrenalectomized patients whose ante.
cedent hypertension had been restored, coincident
with the chronic administration of cortisone jp
doses which averaged about 125 mg/day. The re.
sults were compared with 12 measurements made
in 9 spontaneously hypertensive patients. The
mean diastolic pressures of the 2 groups did not
differ significantly. All patients were maintained
on self-selected diets throughout. Bioassays were
done by the method of Venning (Endocrinology
52: 623, 1953). Reference standards were estab-
lished by assays run with desoxycorticosterone,
The. sodium-retaining factor output of the ad-
renalectomized hypertensive group was about
double that of the spontaneously hypertensive
group (P = 0.05), and equivalent to approximately
20 wg of DCA/hr. Their 24-hr. urinary sodium
excretion was also more than double that of the
spontaneously hypertensive group (P < 0.001)
and averaged 163 mEq/m?/day. This paradox of a
higher sodium output despite a greater excretion
of sodium-retaining factor in the cortisone-treated
group mimics the increased sodium exchange seen
in DCA-hypertensive rats permitted self-selected
salt intakes (GREEN et al. Am. J. Physiol. 170: 486,
1952). (Supported by Los Angeles Heart Assoe.
Grants no. 87 and 88.)

235. Hepatic uptake of radioactive vitamin
Biz in liver disease. GrorGcE B. JERzyY Gass,
LEONARD M. Estn,* Linn J. Boyp AND RoGER
LaucutTon.* Dept. of Medicine and Gastro-
enterology Research Lab., New York Med. College,
New York City.

In order to evaluate the ability of the damaged
liver to take up vitamin By, 35 measurement, of
the hepatic uptake of Co® Bi. were performed in
15 cases of diffuse liver disease. The hepatic uptake
was measured by scintillation counting the surface
of the liver (Arch. Biochem. 51: 251, 1954), follow-
ing parenteral or oral administration of Co By
alone or with potent intrinsic factor preparation.
The data were obtained at various stages of disease
and correlated with liver function tests and clini-
cal picture. The hepatic uptake of orally adminis-
tered Co® Bi. was temporarily abolished in 1 case
of severe virus hepatitis, as well as in terminal
stages of one case of liver cirrhosis and one case of
obstructive jaundice. Out of the remaining 3 cases
of hepatitis, 2 of liver cirrhosis, and 3 of obstruc-
tive jaundice, the hepatic uptake of Co® By» was at
a low level in 6 instances. In 4 out of 5 cases of
metastatic or primary liver carcinoma the hepatic
uptake of Co B,. was normal, both on oral and
parenteral administration. The addition of in-
trinsic factor preparation in 8 out of 9 tests did not
improve the decreased hepatic uptake of Co® Bu.
The impairment of the hepatic uptake of Co Bu,

olume 15

rade in 3
se ante-
incident
isone in
_ The re.
its made
its. The
did not
intained
AVS were
crinology
e estab-
»sterone,
the ad-
s about
rtensive
ximately
sodium
it of the
< 0.001)
\dox of a
xeretion
-treated
nge seen
selected
170: 486,
t Assoc.

vitamin
r GLAss,
» RoGEr
Gastro-
College,

lamaged
ment, of
yrmed in
c uptake
> surface
, follow-
Co” By
aration.
f disease
nd clini-
1dminis-
n 1 case
terminal
e case of
gy 3 cases
obstruc-
12 Was at
cases of
hepatic
oral and
1 of in-
3 did not
Co® Bis.
Co Bu,

March 1956

if present, does not obviously depend on the de-
eased output of gastric intrinsic factor, but on
theimpaired ability of the diseased liver to pick up
or retain vitamin Bis. It appears from these pre-
liminary studies, however, that an advanced lesion
of the liver does not necessarily need to severly
impair the ability of this organ to anchor vitamin
By especially on its parenteral administration.
Supported by Grant-in-Aid A-68 (C3) from the
Natl. Inst. of Arthritis and Metabolic Disease,
PHS, and Merck & Co., Inc., Rahway, N. J.)

86. Paper-electrophoretic analysis of gastric
juice. Grorce B. Jerzy Guass, LOUKIA
STEPHANSON* AND Marityn Ricu.* Gastro-
enterology Research Lab., New York Med. College,
New York City.

The authors have successfully adapted paper-
dectrophoretic technic to quantitative study of
proteins and mucoproteins of gastric juice. After
trials lasting almost 2 yr. with various methods
f concentration and different types of apparatus,
iter testing various buffers, papers, currents,
durations of runs and staining procedures, the
following technic proved most satisfactory:
(entrifuged gastric juice is dialyzed with mechani-
wl stirring against four changes of tap and
listilled water at 4°C for 24 hr. and lyophilized.
Ten mg freeze-dried electrolyte-poor material,
derived usually from 5-10 ml of gastric juice, is
tissolved in 0.5 ml of borate buffer (px 9.0, ionic
trength 0.12) prepared by dissolving 15.3 gm
Ya2B,O7- 10 H.O and 1.24 gm H;BO; in 1000 ml
vater. Samples of 0.06 ml are applied with sample-
driper to the center of 3-cm wide Whatman #1
japer strips and run against same borate buffer in
i Williams hanging strip paper electrophoresis
wil at 0.5 mA/em, 120 v., for 44 hr. Paper strips
ie dried in oven, stained with saturated amido-
lack 10 B solution for 15 min., washed in five
rashings of methanol (9 parts) and glacial acetic
wid (1 part) for 10, 20, and 3 X 30 min. respec-
lvely, dried in open air, and automatically
xanned, traced and integrated in the servo-type
integrating scanner Spinco, provided with special
am Be. From the weight of lyophilized material,
id volume of gastric juice from which it is de-
ived percentual concentration of each component
ak in mg per 100 ml gastric juice is calculated.
ver 80 normal and pathological gastric juices
ere studied with this technic. Excellent resolu-
ion into 7-8 normally reproducible and well dupli-
ted peaks was obtained. Typical paper-electro-
horetic patterns of ‘fasting’, ‘histamine’ and
msulin’ juices of normals and patients with

ious gastric disease will be shown, and their
hysiological and clinical significance will be
iscussed.

AMERICAN PHYSIOLOGICAL SOCIETY 75

237. Toxicity and metabolism of ethyltri-
chloramate (HY-185, 2,2,2-trichloro-l-hy-
droxyethylcarbamic acid, ethyl ester). A. J.
Guazko, L.M. Wotr,* W. A. Ditu* ann J. K.
Weston.* Research Labs., Parke, Davis and Co.,
Detroit, Mich.

Acute oral toxicity determinations gave LD5o
values of 1140+22 mg/kg in mice, and 1460+20
mg/kg in rats. Daily doses averaging 599 mg/kg
given in the diet were well tolerated by rats over a
6-mo. period. Dogs tolerated well single doses of
300 mg/kg daily, administered 5 days/wk. for 1
yr., with no evidence of toxicity or depression.
Monkeys tolerated well doses of 200 mg/kg on a
similar regimen with some sedation, but no evi-
dence of toxicity. Gross and histological examina-
tion of biopsy and autopsy specimens demon-
strated no significant abnormalities due to HY-185
in either species. The metabolism of HY-185 was
compared with that of chloral hydrate (CH) in
animals and in man. Rats receiving 100 mg/kg
oral doses of HY-185 excreted mainly unchanged
drug; while those dosed with CH excreted mainly
trichlorethanolglucuronide (TCE-G), with no sig-
nificant amounts of free drug present. The identity
of HY-185 in rat urine was established by paper
chromatography. Normal human subjects given
0.5 gm doses of HY-185 orally excreted large
amounts of unchanged drug, representing about
50% of the dose. Small amounts of TCE-G were
also excreted. Since HY-185 is just as effective a
hypnotic agent as CH, the observation that a
large part of the drug is excreted unchanged indi-
cates that the intact drug itself has hypnotic
activity.

238. Effect of whole body irradiation on ex-
cretion of sodium and potassium in the rat.
J. L. GLENN* anv W. R. Boss. Dept. of Zoology,
Syracuse Univ., Syracuse, N. Y.

It was the purpose of the present investigation
to determine the effects of sublethal, midlethal and
lethal doses of x-irradiation on the excretion of
sodium and potassium in the rat. Pair-fed controls
were employed to eliminate any changes that
might result from the decreased food intake that
irradiated rats exhibit. X-irradiation was gener-
ated at 140 kvp and 7 ma by a Westinghouse unit
with a Machlett tube. Total body irradiation was
administered in divided doses, 50% of the total
dose being given dorsally and 50% ventrally. The
animals were irradiated in a triangular aluminum
cage which contained three compartments with
perforations on the side for ventilation. Rats re-
ceiving 500 r and 600 r excreted the same amount
of a water load as control animals, but the rate at
which they excreted the water was increased. The
first day postirradiation, polyuria characteristic
of rats is completely eliminated by 24-hr. water

deprivation following exposure. This is offered as
evidence that polydipsia is primary and polyuria
secondary following x-irradiation. The loss of
sodium in the urine of 500 r and 1000 r x-irradiated
rats is not different from that in pair-fed controls.
There is no significant change in serum sodium
during the first 96 hr. following 1000 r. A signifi-
cant increase in urinary potassium occurs on the
first day postirradiation following 500 r, 750 r and
1000 r. This is a real loss and not associated with
decreased food intake or increased urine output.
Serum potassium levels are elevated during the
first 96 hr. postirradiation, being the highest at
2 hr.

239. Inhibition of liver cell division and serum
protein changes induced by dehydration in
partially hepatectomized rat. ANpR& D.
Gutnos (introduced by R. W. CuarKE). Growth
Physiology Lab., Div. of Surgery, Walter Reed
Army Inst. of Research, Army Med. Ctr., Wash-
ington, D.C.

Based on previous findings, a concept of an in-
verse proportiona: relationship between concen-
tration of certain serum components and liver cell
regeneration was formulated (GLINos AND Gry.
Proc. Soc. Exper. Biol. & Med. 80: 421, 1952). To
test further the validity of this concept, restric-
tion of fluid intake was used to increase the con-
centration of serum constituents in rats under-
going liver regeneration after partial hepatectomy.
Under these conditions, liver cell division should
be inhibited. Accordingly, liver mitotic index
determinations and paper electrophoresis of serum
proteins were carried out on 24 pair-fed rats di-
vided into 3 groups and subjected to the following
procedures: J, laparotomy; J7, caudate lobe partial
hepatectomy; III, partial hepatectomy plus fluid
intake restriction. It was found that the mitotic
activity in the livers of group III was totally in-
hibited. Total serum protein was within the
norma range in groups I and JJ, and showed an
increase of 30% in group III. Relative albumin
concentration showed a decrease of 16% in group
I, 23% in group IT and 15% in group IIT. Absolute
albumin concentration showed a decrease of 17%
in group I, 25% in group II and was within the
normal range in group III. Globulin changes were,
in general, opposite to albumin changes.

240. Metabolism of labeled thyroxine in per-
fused rabbit kidney in vitro. Monror §S.
GuitzeR,* Samson Symcuowicz* AND JACK
Gross. Dept. of Biology, Brookhaven Natl. Lab.,
Upton, and Dept. of Anatomy, State Univ. of New
York College of Medicine at New York, Brooklyn.
New Zealand White rabbits were anesthetized

with 25% EtOH, intraperitoneally. Blood was

withdrawn via heart puncture, diluted with an

equal volume of Ringer’s solution, and placed in a

FEDERATION PROCEEDINGS

Volume tj

perfusion reservoir with 2 ug of I*!-labeled thy.
roxine. The left kidney was removed aseptically
and the renal artery and vein cannulated. Th
duration of perfusion ranged from 1.5 to 13 br
Oxygen consumption was observed by the marked
A-V color difference. Urine/plasma ratios of gly.
cose (0.5), sodium (0.8) and creatinine (1.7-25)
indicated that the kidney still retained some fune.
tional capacity. Serial blood samples and kidney
homogenate were chromatographed, counted and
radioautographed. The radioautographs of the
blood samples did not show any triiodothyronin
spots. In the kidney, however, appreciabl
amounts of triiodothyronine were demonstrabk
and more specifically identified in 2-dimensional
chromatograms. All experiments demonstrated
radioactive uptake (2-27%). It could be calculated
that the thryoxine utilization approximated th
triiodothyronine and iodide formation. Thus it was
concluded that thyroxine could be converted to
triiodothyronine in the perfused kidney in vitro,
(Work done under the auspices of the Atomic
Energy Commission and supported in part bya
grant no. A-370-Cz from the Public Health
Service.)

241. Comparison of metabolism of right and
left ventricle and atrium of the rat heart.
Kone-00 Gon anp R. Duncan DALLAM (intno-
duced by Ray G. Daaas). Dept. of Biochemistry,
Univ. of Louisville School of Medicine, Louisville,
Ky.

In confirmation of the reports of others, it has
been observed that the rate of oxygen uptake by
atrial slices is less than by left ventricular slices
from the hearts of normal male rats. In addition,
left ventricular slices were consistently found to
have metabolic rates higher than right ventricular
slices from the same hearts, the average differ.
ence, in 8 hearts, being 24%. The atrial metabolic
rate was approximately the same as that of the
right ventricle. The addition of dl-thyroxine in-
creased the oxygen uptake of all slices, right
ventricle and atrium by about 20%, left ventricle
by about 12%. Oxygen uptake of slices from the
hearts of thyroidectomized animals was less than
that of normal slices; right ventricle by 57%, left
ventricle by 37%, and atrium by 22%. Addition of
dl-thyroxine to these hypothyroid slices increased
the metabolic rate in all by about 40%. (Supported
by grants from the National Science Foundation,
Lederle Laboratories, and American Heart Ass0-
ciation.)

242. Acid-base changes of blood in acute anti-
cholinesterase intoxication. Armanp J.
Goutp, Joun M. WELLER AND GusTAVE FREE-
MAN (introduced by Hans J. Trurnit). Chemical
Corps Med. Labs., Army Chemical Ctr., Md.
Acid-base disturbances following acute intoxica-

Volume {j

beled thy.
aseptically
lated. The
> to 13 hr
the markej
tios of glu.
1e (1.7-25)
some fune.
and kidney
yunted and
rhs of the
othyronine
appreciable
monstrable
'imensional
nonstrated
calculated
imated the
Thus it was
nverted to
y in vitro,
he Atomic
part bya
ic Health

right and
rat heart,
4AM (intro-
ochemisiry,
Louisville,

ers, it has
uptake by
‘ular slices
1 addition,
y found to
rentricular
age differ-
metabolic
hat. of the
Toxine in-
ices, right
t ventricle
3 from the
3 less than
r 57%, lett
ddition of
increased
Supported
yundation,
eart Ass0-

ute anti-
MAND J.
VE FREE-
, Chemical
, Md.

> intoxica-

March 1956

tion of dogs, with alkyl phosphate inhibitors of
cholinesterase are the subject of this investiga-
ti. Lethal doses of parathion and sarin injected
intravenously into anesthetized dogs are followed
by respiratory arrest and severe bradycardia.
These animals terminate in a state of marked
widosis characterized by an average arterial pCO:
devation of 40 mm Hg and a total whole blood
buffer base reduction of 15 mEq/l. in approxi-
nately 30 min. Calculations are according to
finger and Hastings (Medicine 27: 228, 1948).
When elevation of pCO: is prevented by artificial
repiration during intoxication, buffer base falls
st about the same rate and pH at about half the
te. The data, therefore, indicate that acidosis
develops with both metabolic and respiratory
futors, each having a similar degree of effect
won pH. Reduction of buffer base is related, in
part, to the accumulation of lactic acid during
cholinergic intoxication. In the case of asphyxia
by rebreathing alone, acidosis is predominantly
respiratory, buffer base being little affected. With
pisoning it appears that tissues remain hypoxic,
despite maintenance of adequate arterial oxygen
tension by artificial means, secondary to a 4- to
}fold reduction in cardiac rate. Compensation in
wtput for this degree of bradycardia by stroke
volume is untenable.

43. Simultaneous estimation of flow, pres-
sure gradients and ventricular work in
aortic and mitral stenosis. Harry GOLDBERG,
Raupx C. Smita, Luis Vittaca, ASHER WALDOW
anp GEORGE Raser. Hahnemann Med. College
and Hosp., Philadelphia, Pa.

Twenty-five patients with pure aortic stenosis
id 28 with pure mitral stenosis were studied by
imultaneous left and right (combined) heart
catheterization. Right heart catheterization was
performed in the usual way. Left heart catheteri-
ution was performed by the transthoracic ap-
proach by inserting a needle into the left atrium. A
wlyethylene catheter was passed through the
wedle and advanced into the aorta. Pressure
gadients and flows (direct Fick) were thus ob-
tined simultaneously. Valvular flows, areas and
ventricular works were calculated by a modifica-
tion of the Gorlin formulas. In aortic stenosis the
fow was reduced, averaging 163 cc/sec. (85-325).
The left ventricular-aortic systolic pressure
gadients were marked, averaging 60 mm Hg (32-
8). The valve areas were considerably reduced
(.2-1.1 em?). An increase in the total left ventricu-
lr work was observed, averaging 5.2 kg/min. In
titral stenosis the valve flow was diminished,
weraging 103 cc/diastolic sec. (27-261). The left
iitial pressures were consistently elevated (10-39
tm Hg). These correlated well with pulmonary
apillary pressures. Left atrial-left ventricular

AMERICAN PHYSIOLOGICAL SOCIETY

filling pressure gradients were consistently ob-
served, averaging 13 mm Hg (5-28). The effective
right ventricular work was increased, averaging
1.2 kg/min. (14-2.7). The valve areas were de-
creased, averaging 1.0 cm? (0.4-2.6). The pressure
gradients were shown to vary directly as the flow
when the orifice size remained constant in both
mitral and aortic stenosis. As the valve area was
reduced the flow showed a concomitant reduction.
Ventricular work appeared to vary directly with
valve area and with valve flow. Combined heart
catheterization is valuable not only in assessing
the total dynamics in valvular disease but also in
evaluating surgical techniques for correction of
these lesions.

244. Influence of the ‘time factor’ in radiation
effects on biological systems. ANNA GOLD-
FEDER AND Grace E. CuarKe.* Cancer Re-
search Lab., Depts. of Hospitals and of Biology,
New York Univ., New York City.

The length of time during which a given dose of
ionizing radiation is applied to living tissues may
influence the results. Many attempts have been
made by a number of investigators to shed light on
this problem, but no general agreement has been
reached. The discrepancies in results may be at-
tributed to differences in the biological material
studied. Investigations have been undertaken to
establish the time-dose-relationship in the effects
of x-radiation employing genetically uniform ma-
terial. The following schemes of administering a
dose of x-radiation are under study: a) the same
dose delivered at 2 different intensities, b) the same
total dose split into several exposures of shorter
duration but of constant intensity. In part (a) of
this study, inbred mice have been exposed to
whole-body x-radiation in a specially constructed
plastic box, and mouse tumors grown in indigenous
hosts have been x-radiated in situ, the rest of the
organism being shielded. Results obtained so far
have shown that a whole-body dose of 700 r de-
livered at a rate of 8 r/min. exerts a significantly
greater effect than the same dose applied at 765
r/min., as judged by mortality rate and loss of
body, spleen and testicle weights. Similar observa-
tions have been made on mouse tumors. Tumors
x-radiated with 3000 r at 8 r/min. shrank more
rapidly and to a smaller final size than those
tumors x-radiated with 3000 r at 765 r/min.
Further data are being gathered in order to deter-
mine optimum intensity of radiation for single ex-
posures and optimal time intervals between
multiple exposures of the same dose and of con-
stant intensity. (Supported by a grant from the
Natl. Cancer Inst., Natl. Insts. of Health, PHS.)

245. Ion fluxes in dog jejunum: evidence for
active transport of chloride. P. GotpMan,*

W. G. Wa.ker,* K. L. ZrERLER AND W. W.
Scotr. Depts. of Medicine and Urology, Johns
Hopkins Univ. and Hosp., Baltimore, Md.

In anesthetized dogs, by constant perfusion of
Thiry-Vella jejunal loops with various concentra-
tions of NaCl tagged with Na?? and Cl**, a method
has been developed for determining ionic and
water flux across the gut uncomplicated by the un-
certainty of concomitant volume changes within
the lumen. Flux in both directions across the
jejunal wall is calculated simultaneously from
isotope activity and ionic concentration in the
perfused and collected fluid since decrease in
isotope activity is a measure of ion lost from the
gut while ionic concentration differences represent
net ion exchange. For both sodium and chloride a
plot of the ratio of flux leaving the loop to flux
entering the loop is linear with their average ionic
concentration in the gut during transit. For
sodium the two fluxes are equal at a concentration
in the lumen of 138 mEq/].+12 (s.e.m.) as com-
pared with plasmas of 153.3+2.0. For chloride the
two fluxes are equal at a luminal concentration of
149.5411.2 as compared with simultaneous
plasmas of 114.1+1.0. On the assumption that
pertinent fluxes are related to plasma and luminal
concentrations the data support a passive role for
Na flux, but are inconsistent with a passive role
for Cl (P < 0.01).

246. Development of protein granules in the
fat body of Drosophila melanogaster larvae:
normal development. E. D. GOLDSMITH AND
Sot Kramer.* Dept. of Histology, College
of Dentistry and Graduate School of Arts and
Science, New York Univ., New York City.
Twenty-four-hour-old larvae of a Woodbury

wild strain of Drosophila were placed on a yeasted

Pearl’s ‘synthetic medium’ and maintained at a

temperature of 25°+0.5°C. Preliminary observa-

tions hgd disclosed differences in the distribution
and development of certain albuminoid granules
present in the fat body cells of mature normal
larvae as compared with the fat body cells of larvae
treated with an antifolic such as 4-aminopteroyl-
glutamic acid. These albuminoid granules yield a
specifically positive reaction with Millon’s reagent
and the xanthoprotein test and a diffusely positive
reaction with Berg’s ninhydrin reagent. The
granules vary in size from approximately 2.75-5.5
win the mature larvae about to undergo puparium
formation. These granules are not visible in the fat
bodies of Ist and 2nd instar larvae but granules
of this size appear within a period of 6 hr. before
puparium formation (approx. 90 hr. old). During
the prepupal and pupal stages some of these
granules appear to increase further in size, reach-
ing a maximum diameter of 18-19 4. When Dro-
sophilia larvae are reared under conditions of

FEDERATION PROCEEDINGS

Volume 1§

crowding, 200-300 instead of 15 larvae per vial, the
development of these granules in the fat body jg
considerably retarded. From such crowding
studies it appears that these albuminoid granules
are first elaborated in the cells of the fat body
lobes of the thoracic region, since they are present
here in small numbers, even when completely
absent from the fat body lobes in the posterior
abdominal region. (Supported by grants from the
Natl. Cancer Inst. and the American Cancer
Soc.)

247. Relation of adrenal cortex to epineph-
rine conditioning. Maurice S. Go.psten
AND EstTe._E R. Ramey. Dept. of Metabolic and
Endocrine Research, Michael Reese Hosp., and the
Dept. of Physiology, Univ. of Chicago, Chicago,
Tul.

Normal male and female dogs were condi-
tioned, by intravenous injection of successively in-
creased amounts of commercial epinephrine, to
survive more than three times the lethal dose of
this drug (0.8 mg/kg). Tolerance to large amounts
of epinephrine (1.5 mg/kg) was established in one
group of animals by a series of single injections
over a period of 30 days as described by Essex
etal. A second group of dogs acquired tolerance up
to 1.5 mg/kg epinephrine as a result of exposure to
constant intravenous infusion of epinephrine over
a period of 5 hr. on 3 successive days. Epineph-
rine-conditioned animals retained their resistance
to the toxic effects of this drug after the removal
of the adrenal glands. Adrenalectomized dogs that
had been previously exposed to large amounts of
exogenous epinephrine appeared to be more re-
sistant to the traumatic effects of hemorrhage.
These animals maintained a lowered blood pres-
sure (70 mm Hg) for several hours following the
bleeding period and survived the procedure for
24-36 hr., while untreated adrenalectomized dogs
similarly hemorrhaged exhibited a rapid drop of
blood pressure to 30-40 mm Hg and did not survive
for more than 6 hr. Previous conditioning to epi-
nephrine appears to protect the adrenalectomized
animal against some of the injurious consequences
of sympathetic discharge resulting from reflex
adaptation to hemorrhagic stress. In this respect
the response is similar to the protection afforded
the adrenalectomized preparation by pretreat-
ment with autonomic blocking agents.

248. Comparative turnover of cystine-S*® and
lysine-e-C™ in various plasma proteins of
the dog following infusion of doubly labeled
plasma. Patrick D. GoLpswortHy AND WADE
VoLWILER (introduced by CLEMENT A. F1nc#).
Dept. of Medicine, Univ. of Washington, Seattle.
§*-labeled cystine and e-C'4-labeled lysine were

simultaneously administered orally to a donot

of 2
uit
lipi¢

ject
cone
utr
after
must
of si
it tl
prev
liver
been
valu

olume 15

‘vial, the
| body is
crowding
granules
fat body
e present
mpletely
posterior
from the
| Cancer

pineph-
OLDSTEIN
bolic and
)., and the

Chicago,

e condi-
sively in-
hrine, to
1 dose of
amounts
ed in one
njections
by Essex
2rance up
posure to
rine over
Epineph-
esistance
» removal
dogs that
nounts of
more Te-
norrhage.
ood pres-
ywing the
edure for
ized dogs
1 drop of
>t, survive
ig to epi-
.etomized
sequences
ym. reflex
is respect
. afforded
pretreat-

e-S* and
»teins of
y labeled
Np WabE
., Fivcn).
., Seattle.
‘sine were

a donor

March 1956

dog. Twenty-four hours later, doubly labeled
plasma from the donor dog was given intrave-
jously to 2 recipient dogs. Plasma was taken at
intervals from the recipient animals over a period
of 60 days thereafter. Two experiments of this
type were conducted. Fractions I, IT (95% y-globu-
jin) and V were isolated by methods 6 and 9 of
(ohn et al. Fibrin was clotted from fraction I by
the addition of thrombin. Fraction V was resolved
ty starch zone electrophoresis into albumin
(00%) and a-globulin. Aliquots of albumin,
fbrin and y-globulin were oxidized and the S*
ad C' isolated and counted as benzidine sulfate
ud barium carbonate, respectively. Also the com-
bined S*° and C"4 activities in albumin, y-globulin
ad a-globulin were measured in undigested pro-
tin and the contribution of each isotope calcu-
lated from the S* isotopic decay rate. The rates of
les of cpm/mg of cystine-S and cpm/mg of
lysine-e-C occur in an exponential manner and are
identical for a given protein within the same dog.
The half-life of a given protein was found to be
imilar in the 4 recipient animals studied.

49. Biochemical changes in blood and liver
of rats following x-irradiation or nitrogen
mustard treatment. WILLIAM H. GOLDWATER
anp Ceci, ENTENMAN (introduced by Lesuie L.
BenNETT). Biochemistry Branch, U. S. Naval
Radiological Defense Lab., San Francisco, and
Dept. of Physiology, Univ. of California, Berkeley.
Rats have been subjected to large doses (2500 r)
if 250 kv x-radiation and lethal injections of
litrogen mustard, in doses of 4 mg/kg. Serum
lipid concentrations have been compared in nor-
mal fed, normal fasted and the irradiated and in-
jeted rats, up to 3 days following treatment. The
wncentration of all serum lipids decrease in the
utreated fasting rat, but increase in fasting rats
iter being subjected to irradiation or nitrogen
mustard injection. Moreover, the lipid changes are
ifsimilar magnitudes in the 2 types of treatment
it the dosages used. In confirmation of results
previously reported to result from x-irradiation,
lver weights, glycogen levels and lipogenesis have
en found to increase over the normal fasting
vue in rats treated with nitrogen mustard.

4). Lymphatic drainage of large particles.
Frank GoLuAN. Research Lab. and Radioisotope
Service, Thayer VA Hosp., Nashville, Tenn.

The rate of removal of interstitial protein by the
jmphatic system can be measured by external
tinting of subcutaneously injected radioactive
idinated human serum albumin (RIHSA).

Vhereas, crystalloid iodine-131 injected subcu-
imeously attains its biological half-life in 1 hr.,
itakes about 24 hr. for RIHSA to reach this
wint. The rate of clearance can be markedly de-

AMERICAN PHYSIOLOGICAL SOCIETY

layed by changing the solubility of the carrier
protein. The stable I'*' albumin compound was
precipitated by heat at its isoelectric point, by
neutral salts and by heavy metals. In all cases the
lymphatic drainage was delayed. The biological
half-life for precipitates by heat amounted to 1.6
days by salt, to 1.8 days by silver and by zine to
2 days. Antihyaluronidase compounds like Hes-
peridin did not alter the disappearance rate,
whereas adjuvants had a pronounced delaying
effect. A mixture of peanut oil, beeswax and
cholesterol was found to be the most efficient
adjuvant by prolonging the biological half-life of
subcutaneously injected silver precipitated
RIHSA for 3 days. Thus, radioiodinated large
particles can be deposited interstitially for pro-
longed periods of time.

251. Thromboplastin generation: an anatysis
with enzyme thromboplastinase. 8. GoLLUB,
ALEX W. ULIN ANp J. Buack (introduced by M.
E. Maxrie.p). Blood Coagulation Research Lab.,
Hahnemann Med. College and Hospital, Phila-
delphia, Pa.

The purpose of this work was to investigate the
relationship between the thromboplastic compo-
nents of human brain tissue extracts and those of
the Thromboplastin Generation Test. The basic
techniques employed included the use of the en-
zyme thromboplastinase on various plasma, serum
and tissue components and extracts, the adsorp-
tion and elution of the same and their ability to
substitute for each other in the test systems em-
ployed. It has been found that simple mixture of
the 3 components alumina plasma, serum, and
ruptured platelets, in the absence of calcium ion,
generates the equivalent in part of tissue thrombo-
plastic lipid (hereafter called ‘lipid equivalent’).
This is so, inasmuch as both are susceptible to the
destructive action of thromboplastinase and one
may be substituted for the other. Of considerable
interest is the finding that subsequent calcifica-
tion of the mixture of the 3 components causes a
disappearance of the ‘lipid equivalent’ and the
appearance of a different and powerful clot-ac-
celerating activity. This latter activity is not sus-
ceptible to thromboplastinase destruction, thus
differing from tissue thromboplastic activity. The
data support the conclusion that the generation of
human plasma thromboplastin includes the pre-
liminary formation of an active lipid moiety (not
requiring calcium and found preformed in tissue
extracts) which is necessary but not sufficient for
the subsequent production of the powerful ac-
celerator characteristic of the Thromboplastin
Generation Test.

252. Cardiovascular and renal hemodynamic
studies in dogs before and after hypo-

physectomy. M. Jay Goopkinp,* James O.

Davis, Witmot C. Bau, Jk.* AND Rosert C.

Baun. Natl. Heart Inst. and Natl. Inst. of Arthri-

tis and Metabolic Diseases, Bethesda, Md.

The effect of hypophysectomy on cardiovascu-
lar and renal hemodynamic function was studied
in 8 unanesthetized female mongrel dogs. Observa-
tions were carried out before and 3 to 8 wk. after
hypophysectomy in 6 dogs; in addition, 2 animals
were studied only after hypophysectomy. Meas-
urements of cardiac output and renal function
were made within a period of 3 hr. except when
repeat determinations were necessary. Following
hypophysectomy, the cardiac output (direct Fick
method) of 6 dogs decreased from a mean control
value of 3.14 1/min. to 1.85 1/min. Oxygen con-
sumption decreased 27%; changes in arteriovenous
oxygen difference were variable. No consistent
effect of hypophysectomy on femoral arterial pres-
sure was observed. An increase of 66% in total
peripheral resistance occurred. Mean right ven-
tricular and right ventricular systolic pressures
were decreased slightly. Glomerular filtration rate
decreased from an average value for the 6 dogs of
69.8 cc/min. to 27.4 cc/min. while effective renal
plasma flow fell from a mean value of 228 cc/min.
to 81 cc/min. Filtration fraction was unchanged.
The average value for the renal fraction of the
cardiac output was 12.4% before and 7.2% after
hypophysectomy. Renal resistance increased two-
to threefold, an increase which was greater than
that of total peripheral resistance.

253. Kinetics of ultraphagocytesis. RoBEert E.
GossELIn. Dept. of Radiation Biology, Univ. of
Rochester School of Medicine and Dentistry,
Rochester, N. Y.

Unlike exudative leucocytes (polys), macro-
phages isolated from the rabbit peritoneal cavity
extract radioactive colloidal gold from solutions
in vitro. This reaction (ultraphagocytosis) in-
volves “two phases: the reversible adsorption of
gold on the cell surface and the subsequent ir-
reversible removal of surface-bound colloid into
the cell. The latter reaction (called ingestion) ap-
pears to proceed at a rate which is proportional at
any moment to the amount of gold attached to the
cell surface; the latter in turn can be related to the
concentration of extracellular fluid by a simple
adsorption isotherm. At 37°C 3 parameters are
sufficient to characterize the reaction between
gold and a suspension of macrophages, namely an
affinity constant, an adsorption maximum, and a
rate constant of ingestion. Although numerical
values differed markedly among cells of different
exudates, all 3 parameters were estimated in 3
instances. In these suspensions between 2 and 20%
of the surface-bound gold was ingested each
minute (37°C, pu 7.4). Under conditions of surface

FEDERATION PROCEEDINGS

Volume 15

saturation it was estimated that tens of thousands
of gold particles were attached to the surface of ap
average macrophage. This amount of gold, hoy.
ever, occupied less than 1% of the geometric areg
of the cell surface. Although surface saturation
imposed an upper limit on the rate of ingestion,
no practical limit was noted in the capacity of
macrophages to continue the reaction. (Based on
work performed under contract with the U. §,
Atomic Energy Commission at the University of
Rochester Atomic Energy Project, Rochester.)

254. Phosphate transfer in human erythro.
cyte ghosts. D. R. H. Gourtey. Dept. of
Pharmacology, Univ. of Virginia Med. School,
Charlottesville.

It has been reported that human erythrocyte
hemolyzed in the presence of a relatively high
concentration of adenosine triphosphate (ATP)
contain up to 5 times their original ATP concen-
tration when the isotonicity of the medium is
restored (GArDos. Acta physiol. acad. sc. Hung.
6: 191, 1954). This observation has been confirmed
and by modifying the technique it has also been
possible to prepare human erythrocyte ghosts
which contain 5-8 times as much adenosine as the
corresponding control ghosts. The transfer of in-
organic phosphate from a tris-buffered Ringer's
solution into these tissue preparations has been
studied with P*%-labeled ions. Ghosts enriched
with adenosine take up inorganic phosphate at an
accelerated rate and convert it into organic phos-
phorus compounds within the cells. Since these
ghosts have lost the ability to utilize glucose the
energy for the active transfer process must be
derived from the adenosine or its components.

255. Effect of restricted water intake on uri-
nary nitrogen output in man on low
calorie diet devoid of protein. FRANCISCO
GRANDE,* JosepH T. ANDERSON,* HENRY
LONGSTREET TAYLOR AND ANCEL Keys. Lab. of
Physiological Hygiene, Univ. of Minnesota, Min-
neapolis.

Urinary nitrogen was measured in 3 groups of
young men while they subsisted for 16 days on 4
diet of 1000 cal. daily of pure carbohydrate. Sweat
losses were measured during work and sleep. The
physical environment and work regimen were
rigidly controlled and included treadmill walking
costing about 1200 cal. daily. Six men (J) received
900 ml daily, another 6 (JZ) received 1800 ml, and
13 men (JJ) had unlimited water. Food and water
restriction started in J and JT simultaneously after
21 days of control on a good mixed diet; water
restriction continued for 5 days for J and 11 days
for II. Urinary nitrogen loss was inversely related
to the water intake and this difference was not
accounted for by differences in sweat loss. By the

— Cn

tw Qa oS ™& oa

‘olume 15

housands
face of an
1d, how.
tric areg
aturation
ngestion,
pacity of
Based on
he U. §,
versity of
ester.)

erythro-
Dept. of
1. School,

throcytes
vely high
te (ATP)
P concen-
edium is
sc. Hung.
confirmed
also been
te ghosts
ine as the
fer of in-
Ringer's
has been
enriched
rate at an
inic phos-
nce these
ucose the
must be
nents.

e on uri-

on low
“RANCISOO
‘ ‘HENRY
-s. Lab. of
sota, Min-

groups of
days on 4
ate. Sweat
sleep. The
men were
ll walking
’) received
00 ml, and
and water
pusly after
iet; water
nd 11 days
aly related
e was not
ss. By the

March 1956

§th day of, restriction the mean daily nitrogen
losses (urine plus sweat) were 137, 106 and 89
mg/kg for groups I, II and III, respectively. On
days 9-10 the value was 97 mg for IJ and 74 mg for
]II. These differences in N losses are statistically
highly significant; P < 0.001 for the difference
between I and III at day 5 and P < 0.01 for the
difference between IT and III at days 9-10.

256. Renal blood flow and volume from indi-
cator dilution curves. IRvING GREEN (intro-
duced by Purtrp Dow). Depts. of Physiology and
Pharmacology, Med. College of Georgia, Augusta.
The left kidney of morphinized, pentobarbital-

ized, mongrel dogs was exposed retroperitoneally.

Heparin was administered and with the renal

artery clamped for approximately 5 min. the renal

vein was cannulated and its outoflw led into a

jugular vein. Injection of T-1824 was made quickly

into the renal artery at its bifurcation through a

short piece of small polyethylene tubing termi-

nated by a 27 gauge needle. The venous outflow
was temporarily shunted from the ‘jugular path-
way’ into tubes rotating on a kymograph carrying
arecord of tubes, time and injection. The concen-
tration of dye in the blood was determined by
Beckman spectrophotometry of the supernatant
after ethanol precipitation. The inconstancy of
contour of serial washout curves taken at half-hour
intervals, even when the total flow remained con-
stant, attests to the fact that the pattern of blood
flow through the kidney was in flux. While the up-
stroke of the dye-dilution curve was always

smooth, the downstroke almost always showed 1

or more discontinuities, varying from inflections

to full-blown secondary peaks. In some cases 2

well-pronounced peaks occurred in the maximal

concentration and the washout curve assumed a

bimodal appearance. Recirculation is not a factor

since all the dye was collected. Present studies
should elucidate the significance of these results.

(Cardiovascular Research Training Program sup-

ported by grants from the PHS and the American

Heart Assoc.)

47. Cation recovery in red cells after butyl-
alcohol treatment. JAMES W. GREEN (intro-
duced by Marton A. Retp). Bureau of Biological
Research, Rutgers Univ., New Brunswick, N.J.
Human red cells exposed to 0.4 m cold n-butyl

tleohol (made up in 0.16 m NaCl buffered with

phosphate at pu 7.45) for short periods rapidly
lst K and gained Na. This exchange was halted
by diluting the alcohol with cold NaCl solution.

Such butyl alcohol treated and washed cells when

incubated at 37°C in a buffered NaCl medium con-

taining low concentrations of K and glucose ex-
hibited a cation reversal of the sort shown by

Harris (J. Biol. Chem. 141: 579, 1941). With 30-35

AMERICAN PHYSIOLOGICAL SOCIETY

mEq of K/l. of cell suspension, intracellular K
generally increased for as long as 8 hr. In some
cases intracellular Na was decreased during this
period but more generally was maintained at a
nearly constant concentration. Cation recovery
varied with the extent of the initial cation reversal
by the alcohol treatment; the greater the reversal,
the less the subsequent recovery. In no instance
did the cells recover their initial concentrations of
K and Na. When adenosine was added to the in-
cubation medium it was generally found to aug-
ment the glucose effect. In all cases incubation
without glucose or adenosine resulted in the con-
tinued exchange of Na and K with their gradients.

258. Trigemino-vagal connections in the cat.
JouN D. Green, Jacosn De Groot* AND JEROME
Sutin.* Dept. of Anatomy, Univ. of California
at Los Angeles.

Although the occulo-cardiac reflex has been
recognized for many years, and Sherrington has
indicated reflex pathways between the trigeminal
nerve and the vagus, the precise nature of these
mechanisms has been only conjectural. In the
course of a study of supranuclear connections of
the vagus, responses were elicited in the vagus
itself and in its dorsal motor nucleus by stimula-
tion of the cornea, anterior nares and dura mater.
Closely similar responses were obtained by direct
stimulation of the trigeminal ganglion after re-
moval of the forebrain, and these were abolished
by section of the trigeminal root or direct applica-
tion of procaine to the ganglion. The pathway has
been traced along the spinal tract of the trigeminal
and through crossed and uncrossed pathways to
the dorsal nucleus of the vagus. The latency of the
vagal nucleus response is about 1.5 msec, and the
vagus nerve discharge about 7 msec, of which
about 1.5 msec are represented by the conduction
time in the vagus nerve. The properties of this
reflex path have been studied in detail. (Aided by
Public Health Grant B-610.)

259. Physical constants of human skin follow-
ing thermal injury. L. C. GREENE (introduced
by Frep B. Bensamin). Aviation Med. Acceler-
ation Lab., Johnsville, and Dept. of Physiology,
Univ. of Pennsylvania Med. School, Philadelphia.
Direct measurements of skin temperature were

made during thermal irradiation for measured

times according to the techniques described by

Hardy. From these measurements the physical

constants of the skin, thermal conductivity (k),

density (pe), and specific heat (c), were determined

as one value, the product kpc, which is representa-
tive of the thermal inertia of the skin. This value
remains within normal limits during and after
heating of the skin when the pain threshold is not
exceeded and the tissue is undamaged. After the

82 FEDERATION PROCEEDINGS

skin is damaged the kpc increases as much as
7-fold, depending upon the severity of the injury.
The elevation of the kpc is greatest when full
blisters are formed and slightest when only hy-
peremia results. Thus, the changes in kpc may be
attributable to infiltration of fluid into the irradi-
ated site, possible convection within the blister
fluid and changes in local blood flow.

260. Effect of a mercurial diuretic upon oxida-
tive phosphorylation in rat kidney mito-
chondria. Roger L. Greir anp Guorta §.
Jacosps (introduced by FrREepERIcCK GUDER
NATSCH). Dept. of Physiology, Cornell Univ.
Med. College, New York City.

The work of Cohen (Acta physiol. et pharmacol.,

neerl. 3: 512, 1954) indicates that kidney slices from
rats given diuretic amounts of organic mercurials
incorporate less than normal quantities of P*? into
high energy phosphate compounds. Oxidative
phosphorylation in isolated mitochondria was not
demonstrably impaired. After administration of
Hg**-labeled chlormerodrin, large amounts of
mercury have been found in the ‘soluble’ fraction
(Gretr et al. J. Clin. Investigation 35: Jan., 1956).
The present experiments suggest uncoupling of
phosphorylation from oxidation in rat kidney
mitochondria exposed in vitro to amounts of
mercurial diuretic previously found by tracer
studies to be present in renal tissue subsequent to
administration of diuretic doses.

261. Cerebral cortico-subcortical interaction
and insulin. Ropert G. GRENELL AND M.
Wo.sarsut.* Psychiatric Inst., Univ. of Mary-
land, Baltimore.

Previous experiments have indicated that under
‘normal’ circumstances some sort of equilibrium
appears to exist between activity projecting down
from the cerebral cortex and that projecting up
from subcortical centers. The effects of certain
substances such as chlorpromasine and lysergic
acid diethylamide suggest that their primary sites
of action are not the same. The indications are that
chloropromazine affects primarily subcortical
structures concerned with maintenance of psycho-
motor drive and wakefulness. Some observations
with insulin (in cats) also appear to suggest a
primary subcortical effect. Insulin when locally
perfused directly into the cortex demonstrates no
effect whatever. If injected into the systemic circu-
lation, however, the evoked cortical response to
electrical stimulation is markedly enhanced. Pre-
liminary experiments indicate that this increase in
amplitude of the response does not occur when the
cortex has been undercut.

262. Properties and gelation of collagen dis-
solved in neutral salt solutions. JEROME
Gross. Lovett Memorial Labs., Massachusetts

Volume 1§

General Hosp., Dept. of Medicine, Harvard Med,

School, Boston.

Fresh ealf corium, when extracted in 0.1-0.5 M
acetic acid yields very viscous solutions containing
0.1-0.3% collagen. Analysis of dialyzed, lyophil-
ized extract reveals hypro 12.2%, gly 24.0%, tyr,
0.9%, hexose 1%, hexosamine 0.2%, uronic acid
0.1%, pentose 0.1%. When such extracts are di-
alyzed against cold phosphate buffers in the px
and ionic strength range 6.0-8.4, u = 0.1-0.45 or
against cold NaCl in the same ionic strength range,
the solution remains clear and viscous. Viscosity
of such a preparation in phosphate pu 7.6, u = 04
at 5°C is: nsp/C = 42 at C = 0.1%, [nl = 113
(Ostwald). Estimated axial ratio = 180. Single
hypersharp boundary was observed in ultracen-
trifuge. Single, sharp peak was observed by elee-
trophoresis in acid and alkaline ranges. Such
solutions, when warmed to 37°C, form a stiff,
opaque, white gel which, when examined in the
electron microscope, is seen to be composed of
typical striated collagen fibrils with detailed intra-
period fine structure. Gelation of collagen solution
(phosphate pH 7.6, u = 0.4) is completely in-
hibited by 0.4 M urea or 0.2 M arginine. Degree of
inhibition is direct function of urea concentration
and is completely reversible by dialysis. Gelation
is measurably slowed by 0.01 M urea and 0.0005 M
arginine. Potassium iodide in concentrations 0.1
that of the urea present completely reverses such
inhibition. The sensitivity of the system to in-
hibitory agents is strongly influenced by pu, ionic
strength and protein concentration and is signifi-
cantly decreased at physiological ionic strength.
(Assisted by Grant No. A90, C5, Public Health
Service.)

263. Sedimentability of nucleoproteins in
homogenates of perfused rat liver. Pau R.
Gross AND WILLIAM PEARL (introduced by 0.
M. Herr). Biology Dept., Univ. College, New
York Univ., New York City.

Rat livers were perfused with media free of di-
valent cations and containing citrate. Homoge-
nates of such livers, free of nuclei and whole cells,
contain no detectable Ca. Recalcification under
various conditions initiates a series of reactions
which may have significance in the interpretation
of the physical properties of cytoplasm and their
alteration in vivo. Such alterations have been
shown to depend, in invertebrate cells, on re-
actions mediated by divalent cations. Cell di-
vision is accompanied by ‘colloidal’ changes. It is
now possible to study the ‘precipitation’ of nucleo-
protein from rat liver homogenates by Ca with
more rational methods. The precipitation or the
appearance of sedimentability at low RCF
(16,000 X g) in normally nonsedimentable frae-
tions is not instantaneous but proceeds, in the

fa
Le

jecti
thes
and

-y miva

ume 1§

€ 1 Med,

0.5 M
taining
yophil-
7%, tyr.
ic acid
are di-
the pu
0.45 or
1 range,
iscosity
u = 04
= 113

Single
tracen-
ry elec-
;. Such
a stiff,
in the
osed of
d intra-
solution
Lely in-
egree of
itration
relation
.0005 M
ions 0.1
ses such
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Health

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March 1956

presence Of physiological amounts of Ca, over
some hours at 37°C. The kinetics of the reaction
are complex and vary with conditions of initiation.
Ultraviolet difference spectra between controls
and experimentals show that the material acquir-
ing sedimentability is a nucleoprotein. Sedimenta-
bility is a reflection of polymerization or ag-
gregation reactions among submicroscopic nu-
cleoprotein particles of small size. Aggregation is
accompanied by the release of significant numbers
of hydrogen ions and by some binding of Ca by the
sedimented materials. The binding data do not,
however, necessarily support a simple ionic pre-
cipitation hypothesis and might perhaps be inter-
preted in terms of reactions initiated, but not
participated in, by the divalent ion. (Aided by a
grant from the American Cancer Society and by a
faculty summer award to Paul R. Gross from the
Lalor Fndn.)

24. Release of heparin in vitro and in vivo.
ALLAN L. GrosssperG, H. GArctiA-AROCHA AND
Frep McILreatuH (introduced by F. C. Mac-
IntosH). Dept. of Physiology, McGill Univ.,
Montreal, Canada.

The presence of biologically active heparin in
mast-cell rich tissues of the cat and guinea pig, as
well as of the dog, has been established by de-
termination of antithrombin activity antagonized
by protamine, and of metachromatic activity, in
cell-free homogenates. The tissues examined in-
clude cat liver, lung and subcutaneous tissue,
guinea pig liver and lung, and dog liver and lung.
Heparin is present in the bound form in the heavy-
granule fraction of intracellular particles sepa-
rated in isotonic sucrose. It can be released from
hese particles by procedures that release hista-
mine: heating, freezing and thawing, treatment
with hypotonic solutions and treatment with
basic histamine-liberators. In the dog, the release
of heparin into the blood in anaphylactic and
histamine-liberator shock can readily be detected:
in the cat and guinea pig, this is not so. It seems
likely that heparin is released along with histamine
in all 3 species, but that it is only in the dog that
teleased heparin readily gains access to the circu-
lation. (Supported by a grant from the Lasdon
Fndn.)

%5. Thalamic inhibition of subcortically
induced locomotor movements. RoBERT G.
GrossMAN* AND S. C. Wana. Dept. of Physiology,
College of Physicians and Surgeons, Columbia
Univ., New York City.

The mechanism of inhibition of locomotor move-
nents by stimulation of the diffuse thalamic pro-
jection system was studied in 22 cats. Light anes-
thesia was maintained with nitrous oxide-oxygen
id the animal was suspended from a frame.

AMERICAN PHYSIOLOGICAL SOCIETY 83

Rectangular wave stimuli were delivered through
stereotaxically oriented bipolar electrodes. Co-
ordinated locomotor movements were elicited by
stimulation of the field of Forel dorsal to the
mamillary bodies with pulses of 3-5 volts, 100/sec.,
and 2 msec. in duration. Single, reflex steps of the
limb were evoked by manual flexion of the paw
when a reactive hypothalamic point was stimu-
lated at a level sufficient to cause only slight
flexion of the limbs. Stimulation of the nucleus
reticularis of the thalamus with pulses of 4-6
volts, 5-50/sec., and 5 msec. in duration, regu-
larly reduced the frequency and amplitude, or
completely stopped, movements elicited from the
hypothalamus. Inhibition of movement occurred
1-2 sec. after the onset of thalamic stimulation and
persisted for 1-2 sec. after the cessation of stimu-
ation. Complete inhibition of elicited movements
generally could not be maintained for longer than
10-20 sec. During complete inhibition of rhythmic
movements, reflex stepping to flexion of the paw
and extensor and flexor tonus were maintained.
The mechanism of inhibition does not depend on
activation of the projections of the diffuse system
to the parietal and frontal cortex. Subcortically
induced, as well as spontaneous, locomotor move-
ments were inhibited by stimulation of the nucleus
reticularis after removal of the parietal cortex and
the frontal lobes.

266. Effect of low-level x-irradiation on in
vitro oxygen consumption of amphiuma
erythrocytes. R. C. Gruspss, M. A. LEssLerR
AND W. S. Martin.* Dept. of Physiology, Ohio
State Univ. College of Medicine, Columbus.

The oxygen consumption of amphiuma erythro-
cytes, suspended in phosphate-buffered (pH 7.4)
isotonic saline-glucose (70 mg %) solution, was
studied at 25°C, using the Warburg technique.
Curarized animals were bled into heparinized
oxygenated phosphate-buffered iotonic saline. The
pooled erythrocytes were washed 3 times in the
above solution and divided into nonirradiated and
x-irradiated aliquots. Qo. (cu mm/mg dry wt/hr)
determinations were initiated approximately 40
min. after irradiation. The average (25-27 determi-
nations) Qo, values for each experimental (30, 60
and 225 r) and control group were calculated. The
effects of x-irradiation, after 3 hr. respiration, are
reported as percentage changes from their paired
controls. Exposure to 30 r x-irradiation failed to
produce a significant change, but a 2-fold increase
in dosage (60 r) increased the oxygen consumption
15%. Increasing the x-irradiation dosage to 225 r
(Qos 18% greater than control value) did not
markedly enhance the stimulatory response found
at 60 r. These studies indicate that amphiuma
erythrocytes are sensitive to very low levels of
x-irradiation. The failure to find changes in oxygen

84 FEDERATION PROCEEDINGS

consumption proportional to increases in x-irradi-
ation dosage suggests that the dose-response curve
of these cells may be logarithmic rather than
linear in nature. (Supported in part by the Re-
search and Development Division, Office of the
Surgeon General, Department of the Army, under
Contract No. DA-49-007-MD-293.)

267. Initiation of ventricular tachycardia and
fibrillation by rhythmical stimulation of
ventricles of isolated rabbit hearts. LEONARD
GrumBacH (introduced by A. M. Lanps).
Sterling-Winthrop Research Inst., Rensselaer,
N.Y.

The electrical activity of hearts perfused by the
Langendorff method with Krebs-Henseleit solu-
tion was recorded during and after rhythmical
stimulation of the ventricles with rectangular
pulses of varying frequencies. Small serrafine
clamps served as electrodes, the cathode being
placed on the interventricular septum and the
anode on the aorta. Shocks strengths of 100-150 v.
were generally used and the duration varied from
2 to 10 msec. as needed to maintain response at
high frequencies. Ventricular fibrillation could be
initiated by short bursts of stimuli whose fre-
quency was between 11 and 13/sec. Fibrillation
could also be initiated by bursts of steadily in-
creasing frequency if the latter reached 11-13/sec.
With this method, weaker shocks could be used to
maintain response. In most cases the fibrillation
was transient, but in some it persisted for long
periods. Stimulation at frequencies less than
11/sec. never caused any ventricular arrhythmia
either during or after stimulation. However, if
small amounts of procaine were injected into the
perfusion cannula during stimulation at such fre-
quencies, e.g. 5-6/sec., a ventricular tachycardia
occurred which persisted after the cessation of
stimulation. As the procaine washed out, the
tachycardia accelerated and fibrillation occurred
in those Cases where its frequency reached 11-13/
sec. In some cases reversion to a sinus rhythm
occurred before this frequency was reached.

268. Isolation from the hypothalamus of a
substance which stimulates release of ACTH
in vitro. RoGeR GvuILLEMIN, WALTER R.
Hearn, WiituiaM R. CHEEK aND Dwicut E.
HovusHoOLpER (introduced by HessBet E.
Horr). Dept. of Physiology, Baylor College of
Medicine, Houston, Texas.

ACTH secretion by control nonstimulated rat
pituitary is never found (Sayers test) after 48 hr.
of in vitro organ culture life (Proc. Soc. Exper.
Biol. & Med. 89: 365, 1955), whereas saline homoge-
nates of beef hypothalamus (anterior or posterior
region) maintain ACTH secretion up to 8 days of
in vitro survival. Tissues of whole hypothalamus

Volume 1§

(hog, beef) were extracted by Kamm’s procedure
or a modification of it; the end products were ex-
tracted with 90% methanol and chromatographed
on paper (in acetone 60, 2-2’ oxydiethanol 10, aq,
0.05% urea 30). Protopituitrin after methanol ex-
traction and Pitressin were similarly studied. One
fraction, designated as fraction D, common to 4
starting materials (Rf. 95, ninhydrin +) stimu-
lated release of ACTH in vitro (method of Sarrran
AND ScHauy. Canad. J. Biochem. & Physiol. 33:
408, 1955). Active D fractions of hypothalamus and
posterior pituitary have a qualitatively identical
electrophoretic pattern of 5 peaks in 0.5 m HAe,
After elution, only one of these components (DA)
stimulated release of ACTH. Hydrolysis of frac-
tion DA revealed 17 different amino acids; Dé,
therefore, may be a fairly complex polypeptide or
still a mixture. Hypothalamic D is approximately
100 times more active than posterior pituitary D,
w/w. Fraction D from hypothalamus or posterior
pituitary has no vasomotor effects, in contra-
distinction to crude hypothalamic or posterior
pituitary extracts (Rf of vasopressin = 0.4 in
above solvent). Hypothalamic D and posterior
pituitary D expand melanophores of hypophy-
sectomized or normal frogs, show no direct adrenal
stimulating effect, no ACTH potentiation on the
adrenal. No definite evidence of potentiation or
permissive action by arterenol on the ACTH re-
lease by the pituitary could be obtained. (Sup-
ported by grants of Natl. Research Council, Eli
Lilly Research Lab. and Contract AF 41(657)-17,
USAF School of Aviation Medicine.)

269. Urinary excretion of a-ketols on admin-
istration of 9a-fluorohydrocortisone and
Al-cortisone to cats. K. GUNDERSEN AND J.C,
ToucHSTONE (introduced by F. D. W. LuKEns).
George S. Cox Med. Research Inst. and Endocrine
Lab., Hosp. of the Univ. of Pennsylvania, Phila-
delphia.

Daily injections of 9e-fluorohydrocortisone or
A!-cortisone were given subcutaneously to cats.
The urine was collected and extracted with chloro-
form after hydrolysis with 6-glucuronidase and
acid. The a-ketol excretion was measured as blue
tetrazolium-reacting substances according to the
procedure of Touchstone and Hsu (Anal. Chem. 27:
1517, 1955). The administration of 9a-fluorohydro-
cortisone caused a very marked increase in the
urinary a-ketol excretion over control determina-
tions on the same cats. A!-Cortisone gave an in-
creased a-ketol excretion, but 10 mg. of 9a-fluoro-
hydrocortisone administered daily gave higher
excretion values than 50 mg of A!-cortisone. Daily
injection of 10 mg of hydrocortisone gave moder-
ately increased excretion values. a-Ketolic ex-
cretion was still markedly increased 11-13 days
after discontinuation of 9a-fluorohydrocortisone

lic
stil
nor
the
the

eae aa

lume 1§

cedure
ere @X-
raphed
10, aq.
nol ex-
2d. One
on to 4
stimu-
AFFRAN
iol. 33:
nus and
lentical
mu HAe,
ts (DA)
of frac-
is; DA,
»tide or
imately
tary D,
osterior
contra-
osterior

0.4 in
osterior
rpophy-
adrenal
on the
ition or
‘TH re-
|. (Sup-
cil, Eli
657)-17,

admin-
1e and
nD J.C,
UKENS).
ndocrine
|, Phila-

isone or
to cats.
. chloro-
ase and
as blue
g to the
them. 27:
-ohydro-
in the
sermina-
e an in-
x-fluoro-
» higher
e. Daily
> moder-
solic eX-
-13 days
-ortisone

March 1956

injections: Paper chromatographic studies indi-
cate that 9a-fluorohydrocortisone is excreted
mainly as a-ketolic metabolites whereas A!-corti-
gone is mainly excreted as non-a-ketolic metabo-
lites. The studies were made in conjunction withthe
production of steroid diabetes in cats. Preliminary
observations in pregnant or diabetic patients
have not revealed this type or amount of a-ketol
excretion. (Aided by a Traineeship to K. G. from
the Natl. Insts. of Health.)

210. Contrasting effects of bicarbonate and
Diamox, with equivalent alkalinization of
urine, on salicylate uricosuria in man. A. B.
Gurman,* T. F. Yié* ann J. H. Strota. Depis.
of Medicine, The Mount Sinai Hosp., and Colum-
bia Univ. College of Physicians and Surgeons,
New York City.

Renal clearance techniques were employed to
study the mechanisms of salicylate uricosuria. In
6 gouty subjects, sodium salicylate was infused at
rates maintaining plasma salicylate levels >20
mg %. Sodium bicarbonate (15 gm) was then con-
currently injected. The mean premedication con-
trol Curate/Cinulin Of 5.5% rose, due to suppres-
sion of tubular reabsorption of urate, to a mean of
2.6% during salicylate infusion and to 32.4% with
salicylate and bicarbonate; bicarbonate injection
alone (3 cases) only slightly increased Curate/
Ciauliny from a mean of 8.0%-9.7%. This potentiat-
ing effect of bicarbonate on salicylate uricosuria
was related to the degree of alkalinization of the
urine and correlated significantly with an associ-
ated increase in UV free salicylate, from a mean
of 2.92-9.40 mg/min. Such correlations were not
observed in corresponding experiments (3 cases) in
which Diamox (1 gm iv) was used to alkalinize the
urine to the same pH. Diamox decreased salicylate
uricosuria: control Curate/Cinuiin 5.8%; salicylate
alone, 19.0%; salicylate and Diamox, 14.3%.
Diamox alone (8 cases) slightly decreased Curate/
Cinulin, from a mean of 6.4%-5.3%. The Diamox
effects on Curate may be attributable in part to
approximate 10% reduction in Cinyiin. Salicylate,
and salicylate and bicarbonate effects on Curate
were not, however, associated with increase in
filtered urate loads.

271. Positive reflexes as a cause of vicious
cycles in circulatory control systems.
Artuur C. Guyton AND ARTHUR W, LinpsEyY.*
Dept. of Physiology and Biophysics, Univ. of
Mississippi School of Medicine, Jackson.

A positive reflex occurs when an initial stimulus
tlicits a reflex response that adds to the initial
stimulus rather than opposing it. Such reflexes are
normally almost non-existent in the body because
they can cause vicious cycles when the intensity of
the reflex is greater than a certain critical value.

AMERICAN PHYSIOLOGICAL SOCIETY 85

A positive reflex will have a finite stopping point as
long as the response of one cycle of the reflex (y)
is no greater than the initiating stimulus (x) in
accordance with the following formula: total
effect = x + xy + xy? + xy? + --- + xy”. But
when the response of one cycle of the reflex is
greater than 100% of the initiating stimulus the
reflex continues to an infinite effect, thus creating
a vicious cycle. Two positive reflexes studied are
those initiated by 1) pulmonary arterial constric-
tion and 2) bleeding an animal. Either of these two
effects decreases coronary blood flow to the heart.
The decreased coronary blood flow weakens the
heart, which decreases the coronary blood flow
still more. When the pulmonary artery is con-
stricted or when the dog is bled until the systemic
arterial pressure falls to approximately 50 mm Hg,
the reflex response becomes greater than 100% of
the initiating stimulus, and a vicious cycle de-
velops. The heart becomes weaker, coronary blood
flow becomes less, the heart becomes still weaker,
and so forth until the animal dies.

272. Effect of elevation of intraluminal pres-
sure on renal vascular resistance. FRANCIs J.
Happy. Dept. of Environmental Medicine, Army
Med. Research Lab., Fort Knox, Ky.

The right renal vascular system of 40 nembutal-
ized laparotomized dogs has been tested for the
presence of a ‘veni-vasomotor’ or ‘venous-arteri-
olar’ reflex. Renal blood flow was maintained con-
stant by interposing a pump in the renal artery.
Elevation of renal venous pressure from an aver-
age control vaiue of 8 to a final value of 33 was
associated with an immediate rise in renal artery
pressure from 102-142 mm Hg. This 15 mm Hg rise
in gradient (P = <.01) represents active vaso-
constriction since the expected effect of a rise in
transmural pressure in a passive elastic system is
passive vasodilatation with an associated fall in
resistance. The same maneuver in the denervated
kidney caused the gradient to fall on the average
by only 5 mm Hg. Elevation of flow from 25-65
cc/min. was brought about by a 59 mm Hg rise in
artery pressure but only a 3 mm Hg rise in venous
pressure. This was associated with a decrease in
calculated resistance and no significant difference
between pressure-flow curves of the innervated
and denervated kidneys. Therefore, elevation of
arterial and venous pressures evoked reflex vaso-
constriction but preferential elevation of arterial
pressure alone did not. Hence the vasoconstriction
was probably elicited from the veins and likely
represents another example of the venous-arteri-
olar reflex. At least part of the pathway of the
renal reflex is outside of the kidney substance. This
reflex may be involved in the immediate 46% de-
crease in urine flow rate observed when renal
venous pressure was elevated by 22 mm Hg in

86 FEDERATION PROCEEDINGS

intact kidneys (blood flow uncontrolled, nerves
intact).

273. Lipid synthesis by the in vitro perfused
aorta and its perfusate. J. WILFRID HAHN
AND ME vIn A. Nyman (introduced by N. T.
WERTHESSEN). Southwest Fndn. for Research and
Education, San Antonio, Texas.

Experiments have been performed which demon-
strate the ability of a perfused organ to react to
physiologically active substances (vitamins or
hormones) in vitro and thereby produce a charac-
teristic and well-defined pattern of overall lipid
synthesis, thus perhaps simulating the coordi-
nated physiological activities of the organ in the
intact organism. Radioactive lipids, synthesized
from C-acetate (carboxyl labeled), were ex-
tracted at room temperatures with Delsal’s re-
agent from the perfusates and the tissues. Chroma-
tography of these lipids on silica gel resulted in
their separation into well-defined fractions which
on radioactive assay (scintillation counter) pro-
duced characteristic and reproducible ‘lipid
profiles.’ Although all of the radioactive compo-
nents have not been identified chemically, the
profile shows characteristic and unmistakable
changes under experimental conditions. Prelimi-
nary studies show that the blood alone is capable
of extensive synthesis of free cholesterol, phos-
phatides and fatty acids. Esterified cholesterol on
the other hand is apparently not synthesized by
the blood in this system. As an example of the
effect of a vitamin on the lipid profile, para-amino-
benzoic acid (PABA) caused a dramatic increase in
blood phosphatide synthesis, while the cholesterol
fraction was but slightly altered and there was an
apparent depression of synthesis of a fraction
tentatively identified as triglycerides. This effect
has been demonstrated in blood perfused both
through an aorta and through a glass cannula, the
aortal perfusate showing a higher count. The con-
tributiof of aortal lipid synthesis to this system
and the effect of various hormones will be re-
ported.

274. Effect of previous cold acclimatization in
rats shocked by a clamping technique. R. E.
Haist, H. ScuacutTer,* 8. Sip.iorsky,* J. R.
HaMILTON* AND D. G. Baxker.* Dept. of Physi-
ology, Univ. of Toronto, Toronto, Canada.

In rats, shocked by a clamping technique, previ-
ous acclimatization to a cold environment (1°C)
led to a slower fall in body temperature than in
non-acclimatized rats. The survival times were
lengthened and the fall in oxygen consumption
was slower in the previously acclimatized rats as
compared to the non-acclimatized controls. (Aided
by a grant from the Defence Research Board of
Canada.)

Volume 1§

275. Infrared emissivities of some arctic
fauna. H. T. Hamme. (introduced by Mar.
GarET McCoucn). Dept. of Physiology, School of
Medicine, Univ. of Pennsylvania, Philadelphia,
The emissivities of the surfaces of many species

of arctic fauna are very low for visible radiation,

that is, they are white, especially in the winter-
time. If the emissivities of these surfaces were
also low (white) for far infrared radiation, there
would be an important sparing of heat loss to the
upper hemisphere of the animal’s environment,
since the radiant sky temperature may be 30°C to
40°C colder than the air temperature when the
latter is below —40°C. The far infrared emissivities
of the fur and plumage surfaces of 10 arctic species
were measured by comparing the radiant energy
exchange between a radiometer at +20°C and the
fur surface at —20°C with the energy exchange
between the radiometer and a black body at the
same temperature as the fur (—20°C). Since the
emissivity of a black body is 1.00 over the entire
infrared spectrum, the emissivity of the unknown
was, therefore, simply the ratio of the radiometer
deflection when directed toward the unknown to
the deflection when directed toward the black
body. The emissivities of all fur and plumage
surfaces measured were within 2% of 1.00. That is,
in the far infrared all surfaces were black. (Sup-
ported by contract AF-18(600)-1329 with Arctic
Aeromedical Lab., Ladd A.F.B., Alaska.)

276. Increased x-irradiation sensitivity in-
duced in rats by development of resistance
to trauma. JoHN K. Hampton, JR. AND JOHN
W. Mannine, JR.* Dept. of Physiology, Tulane
Univ. School of Medicine, New Orleans, La.

Two hundred fifty-nine male CFW rats, divided
among the 4 groups of the experiment, were ex-
posed to 650 r and 735 r x-rays at 200 kv, 15 ma
with 1 mm Al and $ mm Cu added filtration. Dose
rate was 80 r/min. Compared were controls and
rats made resistant to Noble-Collip Drum Trauma
by a series of sublethal exposures to the trauma.
Irradiation took place 14 days following the last
exposure of resistant rats to conditioning trauma.
Of trauma-resistant (T-R) rats, 73.3% died in 30
days after 650 r compared with 59.4% of controls.
At 735 r the mortality of T-R rats was 95.4% and
controls 75.7%. Under conditions of this experi-
ment the mortality of T-R rats significantly ex-
ceeded that of comparably irradiated controls. A
more extensive experiment, using a range of x-ray
doses at different times following development of
trauma-resistance is sufficiently near completion
to conclude from it confirmation of the previous
finding. These data do not agree with the conelu-
sion by Zweifach et al. (Federation Proc. 14: 168,
1955) that no difference in irradiation sensitivity
existed between resistant and normal rats, but do

=
} aa

ume 1§

arctic
r Mar-
chool of
iphia,

species
liation,
winter-
1S were
1, there
3 to the
nment,
30°C to
1en the
Sivities
species
energy
and the
«change
r at the
nce the
e entire
nknown
iometer
10Wn to
e black
plumage
That is,
<. (Sup-
1 Arctic

ity in-
istance
(D JOHN
, Tulane

divided
were @X-
y, 15 ma
on. Dose
rols and
Trauma
trauma.
the last
trauma.
ied in 30
controls.
4% and
3 experi-
intly ex-
ntrols. A
of x-ray
pment of
mpletion
previous
e conclu-
14: 168,
nsitivity
s, but do

March 1956

agree witl: them in presenting evidence that
trauma-resistance probably involves changes in
physiological systems other than those most
significantly affected by irradiation.

277. Influence of the adrenal on distribution
and excretion of electrolytes. KENDRICK
Hare AND Ruts S. Hare.* Depts. of Pharma-
cology and Pediatrics, Univ. of Alabama, Birming-
ham.

Dogs were adrenalectomized and maintained on
oral cortisone until 24, 48, or 72 hr. before the ex-
perimental procedure. Two types of experiment
were performed: bromide space determinations
and osmotic diuresis with one molar mannitol by
vein. Withdrawal or administration of cortisone
did not affect bromide space in adrenalectomized
or normal animals. During osmotic diuresis in
mild adrenal insufficiency the course of the osmotic
U/P was essentially normal. Although percentage
excretion of filtered water, solute, sodium and
potassium increased, absolute excretion was
similar to the normal or decreased because of
diminished filtration rate. Serum sodium and chlo-
ride concentrations fell more during the diuresis
than those in the normal dog and did not rise as
rapidly after the infusion stopped. In more severe
adrenal insuficiency, the osmotic U/P was initially
lower and fell to one or less during osmotic diuresis.
Absolute excretion of all substances studied was
greatly decreased, although the percentage ex-
cretions were further increased. Filtration rate
was reduced to about 20% of normal. In spite of
diminished excretion, serum sodium and chloride
concentrations fell as much as, or more than in
mild adrenal insufficiency. Serum electrolyte
changes during osmotic diuresis show that the
adrenalectomized animal is less able than the
normal to maintain serum sodium and chloride
concentrations in the face of rapid renal loss. In-
creased renal excretion does not account for the
greater changes in adrenal insufficiency, and sta-
bility of the bromide space suggests that altered
distribution is not responsible.

78. Resting turnover rate of prothrombin.
P. V. Harper,* ELLEN Catvary* anp J. GArR-
rotr ALLEN. Dept. of Surgery, Univ. of Chicago,
Chicago, Ill.

The rapid disappearance of prothrombin from
the circulating blood following hepatectomy,
acute poisoning of the liver, or dicumarolization
suggests that it is utilized at a very rapid rate.
Restoration of prothrombin activity in the plasma
following plasmapheresis and following restora-
tion of vitamin K deficiency indicates an equally
tapid rate of formation. Estimates of prothrombin
turnover time based on such measurements vary
from 6 to 24 hr. Obviously measurements made on

AMERICAN PHYSIOLOGICAL SOCIETY 87

the above described systems were under grossly
distorted circumstances. In the present study an
attempt has been made to determine prothrombin
turnover in a relatively undisturbed system.
Tagged prothrombin was produced in the plasma
following intravenous administration of carbon"
glycine at tracer level to dogs. The prothrombin
was isolated from the plasma by the methods of
Seegers and the disappearance of radioactivity
from this fraction as a function of time was meas-
ured. The specific activity-time curves in 3 ani-
mals were practically identical, indicating a turn-
over time of about 3 days which was constant over
3-4 half lives. While the interpretation of the
slopes of specific activity-time curves as represent-
ing turnover rates is not strictly correct, it is ap-
proximately so, and the turnover indicated in
these experiments is significantly slower than that
estimated from physiologic data. Turnover times
were likewise estimated from the disappearance
curves of the albumin, globulin and fibrinogen
fractions of the same blood samples. The turnover
rates of the clotting components were much faster
than those of the other plasma protein fractions.

279. Atrophy in denervated muscles; effect of
muscle length. A. Sipney Harris AND ALVIN
M. Cortuar.* Dept. of Physiology, Louisiana
State Univ., New Orleans.

In each of 9 dogs the tibial and common peroneal
nerves of one leg were sectioned above the knee.
After periods ranging from 7 to 12 wk. the tibialis
anterior and gastrocnemius muscles were carefully
excised from denervated and control limbs. Com-
parison of weight of each denervated muscle with
the corresponding control from the unoperated leg
was made and the weight difference was considered
a measurement of atrophy. Weight loss was mark-
edly greater in the tibialis muscles (av. 52%, range
37-66) than in the gastrocnemii (av. 22%, range
10-32). The denervated limb often was used as a
prop in standing or walking, placing some stretch
upon the gastrocnemius and passively shortening
the tibialis. To test the suggestion that lengthen-
ing provides some protection against atrophy, 3
additional groups of dogs were studied with casts
applied to the denervated limbs: 1) with joints in
‘neutral’ position; 2) plantar flexed, with short-
ened gastrocnemius and lengthened tibialis; 3)
dorsiflexed, with lengthened gastrocnemius and
shortened tibialis. In groups 1 and 3 atrophy
was greater in tibialis, the difference being more
pronounced in group 3. In group 2 there was a
slight reversal, the average degree of atrophy
being greater in gastrocnemius. The markedly
greater degrees of atrophy in tibialis in the un-
casted and in group 3 were abolished by plantar
flexion. Atrophy in the long digital extensor was
inconsistent except that it was greater in casted

88 FEDERATION PROCEEDINGS

than uncasted limbs. (Aided by medical student
fellowship from the Natl. Fndn. for Infantile
Paralysis to Alvin M. Cotlar.)

280. ‘Fusion’ beats in experimental myo-
eardial infarction. A. SipNEY HaRRIS AND
Ricuarp A. Liprax.* Dept. of Physiology,
Louisiana State Univ., New Orleans.

The occurrence of fusion beats of the Wolff-
Parkinson-White configuration in association with
myocardial infarction has clinical interest and
may contribute to the understanding of mecha-
nisms of anomalous A-V transmission of excita-
tion. Review of electrocardiograms of a large
series of experiments with myocardial infarction
in dogs has revealed a number of records exhibiting
short P-R intervals of almost constant duration
followed by prolonged QRS complexes, and T
waves in a direction opposite to QRS. Such con-
figurations have not occurred often, and then com-
monly were intermixed with cycles exhibiting
normal excitation and conduction relationships
and sometimes with ventricular ectopic beats. A
small portion of the boundary of the infarct some-
times is overlaid by the left auricular appendage,
thus providing physical conditions under which
excitation of boundary cells by the auricular
action potential might take place. Some of the
apparent fusion beats that occurred spontaneously
were of forms that were generally similar to con-
tours of experimentally produced fusion beats
arising in this region. Other spontaneous ‘fusion’
beats in infarction had configurations that suggest
ventricular pre-excitation in the upper right ven-
tricular border of the infarct, in the region of the
interventricular septum. In other records are
found evidences of complete functional A-V dis-
sociation with P waves occurring at continuously
varying relationships to QRS of ventricular origin
without apparently affecting the form of QRS.
(Supported in part by Grant H-1109(C3), National
Heart Insts., Natl. Inst. of Health, PHS.)

281. Influence of certain indoles on cerebral
synaptic transmission. E. Ross Harr,*
Tuomas W. LaneFitt* AND AMEDEOS. MARRAZZI.
Neurology Branch and Clin. Research Div.,
Chemical Corps Med. Labs., Army Chemical Ctr.,
Md.

Observations from this laboratory on the cere-
bral synaptic inhibitory action of adrenaline have
been supplemented by data on the influence of
related substances on the optic cortical response
evoked by activating the transcallosal pathway in
the nembutalized cat. Of great interest has been
the synaptic inhibitory action of serotenin, which
is about 20 times as potent as adrenaline in the
same animal. Since serotonin occurs naturally in
the brain, it is a serious contender for the role of

Volume 1§

cerebral neurohumoral synaptic inhibitor. Synap.
tic inhibition can result in abnormal patterns of
activity either directly or through the release of
downstream neurons from the influence of normal
controlling pathways. It was therefore of interest
to think of these mechanisms in connection with
the production of mental and behavioral disturb-
ances. Adrenochrome and bufotenine have both
been held responsible by various workers for cere-
bral dysfunction and mental manifestations. In
addition, adrenochrome is a first oxidation product
of adrenaline, converting it to an indole, and there-
fore relating adrenaline to serotonin, while
bufotenine is a dimethyl] analog of serotonin. The
experiments show that both are capable of produe-
ing synaptic inhibition, but adrenochrome only in
unreasonably large doses. These findings suggest
that a disturbance of the metabolism of serotonin
or some such substance or of the threshold of cere-
bral cells to it might sufficiently disturb the
balance of neurohumoral excitatory and inhibitory
influences at cerebral synapses to be responsible
for natural mental disturbances such as schizo-
phrenia.

282. Potassium effect on resting membrane
potential of muscle in the intact rat. J. C,
Harvey,* J. L. Linrenrua, JR.! ann K, L,
ZIERLER. Depts. of Environmental Medicine and
Medicine, Johns Hopkins Univ. and Hosp.,
Baltimore, Md.

Resting membrane potentials (E,) were meas-
ured with KCl-filled glass micro electrodes intro-
duced into single muscle fibers of the gastroc-
nemius in the intact rat. Local ionic concentra-
tions were altered without disturbing blood
circulation by infusion at the bifurcation of the
aorta through a catheter inserted into the contra-
lateral femoral artery. Concentrations of serum
Kt and Na* were measured in jugular venous
blood and were undoubtedly substantially lower
that those reached in the extracellular environ-
ment of the gastrocnemius. In 500 measurements in
49 rats not infused E, was 80.2+7 mv. In 25 of these
rats serum K* was measured and E, was found to
vary inversely with serum K*. E, in 15 impale-
ments was maintained for } to 2 hr. Intra-arterial
infusion of 3 m NaCl (0.8 ml in 10 min.) did not
disturb E,. Intra-arterial infusion of 3 m KCl
(0.8 ml in 10 min.) caused a prompt fall in E, by
15-20 mv. This occurred before there was a de-
tectable rise in jugular venous K* concentration.
(Supported by contract with ONR.)

283. Methods for measurement of spinal fluid
gas tensions. RIcHARD F. HARVEY AND GEORGE
A. Saxton, Jr. (introduced by Victor GUILLE-

1 Deceased.

ume 1§

Synap-
erns of
ease of
normal
nterest
m with
listurb-
re both
or cere-
ons. In
yroduct
i there-

while
in. The
produc-
only in
suggest
rotonin
of cere-
irb the
1ibitory
yonsible
schizo-

nbrane
t. JG,
» K. L,
cine and

Hosp.,

e meas-
8 intro-
gastroc-
ncentra-
z blood
1 of the
- contra-
f serum
venous
ly lower
environ-
ments in
of these
found to
impale-
-arterial
did not
om KCl
in E; by
as a de-
atration.

ral fluid
. GEORGE
GUILLE-

March 1956

MIN, JR.)+, Dept. of Preventive Medicine, Univ.

of Illinois College of Medicine, Chicago.

The partial pressure of O2 and COz in the spinal
fuid of human subjects was investigated in order
to approximate the tensions of these gases in the
nervous system in various disease states. Spinal
fuid was sampled from the lumbar region in 11
neurological patients and 5 patients without
organic neurological disease. Oxygen tensions were
measured by the polarographic method using a
falling drop mercury electrode. The current pro-
duced by the spinal fluid at 0.5 volts was measured
and compared with a calibration curve derived
from current readings produced by bubbling gases
of known Oz concentration through the spinal
fuid. Carbon dioxide tensions were measured by
we of a pH meter. The pH reading obtained ini-
tially on the spinal fluid was compared with a
calibration curve derived from pH readings pro-
duced by bubbling gases of known CO: concen-
trations through the spinal fluid. Spinal fluid Po,
ranged from 6 to 31 mm Hg; central nervous sys-
tem trauma had occurred in the 2 patients with
readings below 10. The ‘normal’ cases ranged
from 22-31 mm Hg, with a mean of 27. Spinal
fuid Peo, ranged from 31-75 mm Hg, being rather
consistently related to alveolar Pco.. In 8 cases
of simultaneous studies the Pcs¥rco, was 4-23 mm
Hg above the Paco;, the mean difference being 12.
When spinal fluid was stored anaerobically at
room temperature or at 1-5°C the Po, dropped
blow 10 mm Hg consistently though the Pecos
did not change significantly.

4. Function of blood-perfused dog kidney
at low temperature. RopNEy B. Harvey
(introduced by Virainta D. DAvENpoRT).
Dept. of Physiology, Univ. of Utah, Salt Lake
City.

Dog kidneys were perfused with arterial blood
fom heparinized dogs. Polyethylene tubing was
wed to conduct the blood through water baths
0 that the temperature of the perfusing blood
ould be changed independently. Urine was col-
leted from the isolated kidney ureter and from
the perfusor’s bladder. The variables measured
were perfusion pressure, isolated kidney blood
fow, blood temperature, urine flow and urine
wncentrations of sodium, potassium and creati-
tine. Cooling the isolated kidney to 5°C resulted
in large changes in urine composition and small
thanges in urine flow and blood flow. U/P ratios
sodium, potassium and creatinine approached
nity. Upon rewarming to 38°C the isolated kidney
tine composition returned to approximately
hat of the perfusor’s. In addition to temporary
uppression of active tubular transport at 5°C
hese results suggest that either there is little if
uy tubular water reabsorption or that tubular

AMERICAN PHYSIOLOGICAL SOCIETY

fluid containing creatinine and other substances
at plasma concentrations is reabsorbed. The
temperature characteristics of tubular reabsorp-
tion were found to be different for the substances
studied.

285. Influence of insulin and cortisone on
glucose utilization. F. X. HAusBERGER AND
B. C. HausBerGER (introduced by S. A. D’An-
GELO). Jefferson Med. College, Philadelphia, Pa.
In vitro oxidation of, and lipogenesis from, radio

glucose was determined in adipose tissue of nor-

mal, insulin and cortisone treated rats. Cortisone
administration (5 mg/day for 5 days) produced
in liver slices only a slight decrease of glucose
oxidation. Insulin administration markedly en-
hanced glucose oxidation in both tissues which
was not significantly influenced by simultaneous
cortisone treatment. Cortisone did not change the
normally low hepatic lipogenesis but considerably
diminished the increase seen after insulin adminis-
tration. Cortisone treatment reduced lipogenesis
of normal adipose tissue but not the enhanced
lipogenesis produced by insulin injections. In
vitro glucose utilization was normal in either tissue
of severely steroid diabetic guinea pigs, whereas
in alloxan diabetic rats lipogenesis is abolished
and oxidation diminished. These results were
compared with in vivo observations. Steroid
diabetic guinea pigs utilize normal amounts of
carbohydrates as determined by the classical
method of balancing carbohydrate intake and new
formation with glucose loss. Such animals show
mild obesity, considerable hypertrophy of the
islets (suggesting increased insulin secretion) and
increased food intake and gluconeogenesis. Rats
receiving insulin for 2 wk. show approximately
twice the amount of adipose tissue found in con-
trols, without changing its normal fat, water and
nitrogen content. Simultaneous cortisone adminis-
tration (2.5-5 mg/day), although preventing gain
of body weight, does not suppress this response
of adipose tissue to insulin. (Supported by PHS
Grants A-615 and A-345.)

286. Absence of an antihypertensive effect
of bilateral frontal lobectomy or leucotomy
in dogs hypertensive from bilateral carotid
sinus constriction. Epwarp W. HawTHoRNE
AND CLARENCE 8S. GREENE.* Depis. of Physiol-
ogy and Surgery, Howard Univ. College of Medi-
cine, Washington, D. C.

Bilateral carotid sinus constriction (BCSC)
was produced in 11 dogs by the technique of
Wakerlin and Associates (Circ. Res. 2: 416, 1954).
The carotid sinus clamps used were as described
by Wakerlin and obtained through his courtesy.
Ten of the 11 dogs so constricted developed a
chronic hypertension. The one dog in which hyper-

tension did not develop weighed 14.0 kg and had
aortic insufficiency at the time of BCSC. One of
our interests in such animals is to determine
whether or not there are certain cortical areas
which might play a pathogenetic role in hyper-
tension that follows BCSC. We sought first to
evaluate the antihypertensive effect of bilateral
frontal lobectomy or leucotomy in dogs with
hypertension following BCSC. Our present find-
ings do not permit the possibility that bilateral
frontal lobectomy in such dogs has a chronic anti-
hypertensive effect.

287. Fibrinolytic changes in the dog in re-
sponse to protamine sulfate as demon-
strated by a new and rapid method. R.
Heinrich AND E. Noe (introduced by JAN
NysBoer). Anemia Research Lab. and Cardio-
vascular Research Dept., Harper Hosp., Detroit,
Mich.

The fibrinolytic system of the dog is the subject
of study for the purpose of investigating possible
etiological factors concerned with the bleeding
diathesis observed following surgery and shock
as described by Tagnon and Coon (Am. J. Med.
Sciences 211: 88, 1946; S. G. & O. 95: 717, 1952).
Canine fibrinolysin has long been considered re-
fractory to activation by streptokinase. Hitherto
up to 5 days were required for evaluating such
activity. A rapid method requiring less than
10 min. to determine changes in fibrinolytic ac-
tivity in the dog using streptokinase has been
developed. Jaques has reported that protamine
sulfate in excess of 10 mg/kg will activate fibri-
nolysin (Br. J. Pharmacology 4: 135, 1949; Proc.
Soc. 66: 326, 1947). We have demonstrated definite
changes in fibrinolytic activity by administering
2.5 mg/kg i.v. protamine sulfate to normal dogs
and to heparinized dogs where the heparin-
protamine ratio was 1:1 or 1:1.28, the commonly
used therapeutic range. We have also found a
change‘in fibrinolytic activity during the course
of cardiac surgery performed on dogs. Control
studies on dogs anesthetized with intravenous
pentobarbital (20-30 mg/kg) showed no significant
fibrinolytic activity by our method. The effect
of an inhibitory factor as a possible etiological
agent for the changes observed in the fibrinolytic
meciianism has been considered and an evaluation
of this is in progress based on determinations of
the active and total fibrinolysin and antilysin.

288. Reticuloendothelial function and anti-
reticulocytotoxic sera. J. H. HEuuER, D. A.
Bororr AND R. M. MeEtrer.* Divs. of Physiol-
ogy and Microbiology, New England Inst. for
Med. Research, Ridgefield, Conn.

Impressive results and equally impressive
failures have been accredited to antireticulocyto-
toxic sera (ACS). The claims and counter-claims

FEDERATION PROCEEDINGS

Volume 1§

relating to reproducibility of results apply to
both the experimental and clinical fields. It ig
difficult to evaluate many of the reports because
quantification as to what these sera specifically
are or do has been notably absent. The maip
method for assay of presumptive activity is via g
complement fixation test. Methods are now avail-
able which permit the determination of the effect
of substances such as ACS upon certain phases of
the activity of the reticuloendothelial system
(RES). One of the most sensitive of these tests is
that utilizing the phagocytic ability of RE cells,
Antireticulocytotoxic sera were made by the
injection of a saline extract of mouse spleen intra-
venously into rabbits. Activity was assayed by
complement fixation. Sera which met the criterion
of activity by this method of test were injected
into mice in ‘stimulatory’ and in ‘inhibitory’
doses. The animals were then subjected to quanti-
tative evaluation of phagocytic activity of their
RE systems. RE function in individual organs was
specifically evaluated. Neither the so-called
inhibitory or stimulatory injections of ACS showed
any demonstrable change in over-all RE activity.

289. High sensitivity radiometer for skin
temperature measurements during ex-
posure to thermal radiation. EK. HENpiER*
AND J. D. Harpy. Dept. of Physiology, Univ.
of Pennsylvania Med. School, Philadelphia.

A radiometric method for measuring rapid
changes in skin and surface temperature during
exposure to thermal radiation (far infrared) is
described. A specially designed revolving metal
disk chops both the incident radiation falling
upon the surface and the emitted radiation from
the surface 13 times/sec. The emitted radiation
is detected and recorded by the thermocouple
detector and amplifier of a Perkin-Elmer infrared
spectrometer connected to a Brown recording
potentiometer. A Leslie cube is used for calibra-
tion purposes. Thermocouples are used to measure
Leslie cube, disk and detector thermocouple tem-
peratures. The method described allows for accu-
rate measurements of surface temperature without
actual contact with the surface during long or
short exposure durations. Surface temperature
changes can be continuously measured within
0.005°C. Measurements of the kpe product (k =
thermal conductivity, p = density, c = heat
capacity) can be made for bare unblackened skin
using the above method under various environ-
mental conditions. In addition, the method is
sufficiently sensitive for investigations of skin
temperature changes accompanying — thermal
sensations.

290. Effects of cooling of cortical respiratory
area on exercise hyperpnea in the dog.
Joun P. Henry* ann W. V. Wurrexorn. Depl.

4. B.

(res

tate
to (
gat
clea

eas ta a

lume 15

pply to
s. It is
because
cifically
1e main
is via a
W avail-
1e effect
hases of
system
- tests is
-E cells.
by the
n intra-
ayed by
riterion
injected
\ibitory’
. quanti-
of their
rans was
0-called
} showed
activity.

yr skin
ng ex-
ENDLER*
y, Univ.
ghia.
g rapid
e during
‘ared) is
1g metal
1 falling
ion from
-adiation
nocouple
infrared
ecording
-calibra-
measure
ple tem-
for accu-
» without
- long or
perature
1 within
ict (k =
= heat
»ned skin
environ-
iethod is
- of skin
thermal

piratory
he dog.

RN. Depl.

March 1956

of Physiology, Univ. of Illinois College of Medi-

cine, Chicago.

The significance of cortical contribution to
hyperpnea of exercise has long been debated.
Attempt was made to evaluate the role of this
factor by cooling of respiratory area of orbital
cortex in 20 experiments on 13 Nembutal-ure-
thane anesthetized dogs before, during and after
dectrically induced exercise of hind limbs. Orbital
cooling during eupnea produced 10% decrease in
respiratory rate with questionable reduction of
minute volume. Cooling induced during exercise
with mean metabolic rate ratio of 2.2 resulted in
reduction of mean ventilatory volume from 4.85
l/min. to 4.37 1/min. Difference is highly signifi-
cant statistically and represents 20% of the
exercise hyperpnea under these conditions. Cessa-
tion of cooling was followed by return to pre-
cooling levels. Results indicate that cortical
impulses contribute to exercise hyperpnea even
under conditions of anesthesia and low levels of
exercise and suggest a quantitative approach to
evaluation of this factor under more physiologic
conditions.

91. Renal clearance in the alligator. THomas
HERNANDEZ AND Rouanp A. Couxson.* Dept.
of Biochemistry, Louisiana State Univ. School
of Medicine, New Orleans.

Creatinine and thiosulfate clearances were
determined and found to agree substantially
with the values reported by Shannon for inulin.
Plasma levels varied from 0.2 to 1.6 mg% for
creatinine and 10 to 110 mg% for thiosulfate.
From clearance studies, glomerular filtration
trate varied from 0.05 cc/K/min. in dehydration
to 0.2 ce/K/min. in extreme hydration. The alli-
gators used weighed from } to 3 kg. Creatinine
tlearances were compared with those of K, SO,,
PO,, urea, lactic acid and exogenous glucose. At
high plasma levels, urea, lactic acid, glucose and
sulfate clearances averaged about 50% of that for
weatinine. Na and Cl are almost completely
absorbed but K clearance varied from almost
wro to 50% of the creatinine clearance. Since
PO, clearance is 3-fold that of creatinine, it is
probable that PO, is secreted by the renal tubules.
The ‘osmolar clearance’ varied from 3.0 cc/K/day
innormal alligators to 20 ce/K/day following salt
lading. Nonelectrolytes such as urea and glucose
depressed the excretion of the normal urinary
dectrolytes without affecting their relative ratios.
Since the alligator cannot concentrate his urine
tbove that of the plasma, all compounds excreted
nust find room below the ‘osmotic ceiling.’ (Sup-
ported in part by PHS Grant No. H-2062.)

2. Functional role of brain stem reticular
system in salivary conditioned response.
R. HerNnaANnpDEz-Peon, H. Brust-Carmona,

eer,

AMERICAN PHYSIOLOGICAL SOCIETY 91

E. Ecxuavus, E. Loprz-Menpoza anp C. AL-

COCER-CUARON (introduced by J. J. IzqureRpo).

Dept. of Physiology, School of Medicine, Univ.

of Mexico, Mexico, D.F.

One of the basic problems in the investigation
of neurophysiological mechanisms of learning is
that of detecting he locus for plastic association
within the central nervous system that is re-
sponsible for conditioning. Since apparently
cerebral cortex is not essential for the establish-
ment or retention of simple conditioned responses
(Hiuearp, E. anv D. Maraquis. Conditioning and
Learning, New York: Appleton, 1940), subcortical
mechanisms must be necessary, at least for
the plastic changes involved in the most simple
types of learning. Conditioned salivation was
established to visual and tactile stimuli, using
inhalation of ether as unconditioned stimulus in
cats with the parotid gland duct cannulated. Once
abundant conditioned salivation was established,
restricted lesions were made by electrolysis in a
number of subcortical structures (mesencephalic
reticular formation, septum, medial thalamic
nuclei, superior colliculus) by means of electrodes
estereotaxically placed. Among all of these lesions,
only those in the mesencephalic reticular forma-
tion, never resulting in coma, abolished or greatly
reduced in these awake animals the conditioned
salivary response (falls from 20 drops of saliva/
min. before lesion to 0 or 1.5 drops/min. after
lesion were repeatedly observed). On the other
hand, unconditioned salivation was unaffected,
or even enhanced, 1 wk. after the mesencephalic
lesion. These experiments suggest that similarly
to what we have reported in connection to habitua-
tion (negative learning) (Federation Proc. 14:
71, 1955), the simplest type of positive learning
(classical conditioning) also requires functional
integrity of the brain stem reticular system.

293. Photic potentials in the visual pathway
during ‘attention’ and photic ‘habitua-
tion.’ R. HERNANDEZ-PEon, C. GuzMAN-
Fiores, M. Atcaraz AND A. FERNANDEZ-
Guarpio1a (introduced by J. J. Izqurerpo).
Dept. of Physiology, School of Medicine, Univ.
of Mexico, Mexico, D.F.

Photically evoked potentials were recorded with
an oscilloscope or an electroencephalograph in
unanesthetized unrestrained cats with implanted
electrodes in the visual pathway (optic tract,
lateral geniculate body, visual cortex) and mesen-
cephalic reticular formation, during the processes
of attention and habituation. When the animal’s
attention was called by and focused upon acoustic
or olfactory stimuli, partial or total abolition of
photic potentials all along the visual pathway
and reticular formation occurred. The scale of
depressions thus observed, seemed proportional

92 FEDERATION PROCEEDINGS

to the degree of attention. A similar blockade
occurred when attentive behavior was elicited by
stimulating the brain stem reticular formation.
When the photic stimulus was regularly presented
every 5 or 8 sec., potentials from the lateral
geniculate and visual cortex gradually diminished
(habituation), but those from the optic tract
remained unchanged. This condition was observed
to persist as long as 24 hr. Dehabituation, as
judged from recovery of thalamic and cortical
potentials, appeared after a variable period of
rest. It was also elicited by intense extraneous
acoustic or photic stimuli. Inhibition of lateral
geniculate potential was also released during
nembutal anesthesia. It is concluded that during
attention afferent blockade is exerted by inhibi-
tory centrifugal fibers to the retina, and that by
photic habituation sensory inhibition occurs at
the lateral geniculate. Apparently, this inhibitory
influence proceeds from the brain stem reticular
system.

294. Capillary counts in different organs of
warm and cold acclimated white rats.
O. Herovux (introduced by J. 8. Hart). Div.
of Applied Biology, Natl. Research Labs., Ottawa,
Canada.

Capillaries were counted on benzidine stained
cross-sections of leg muscles (soleus, gastroc-
nemius, plantaris), ears, liver and heart of rats
killed with ether after acclimation to 30°C. (Ja),
acclimation to 30°C and exposure to 6°C for 2 hr.
(Ib), acclimation to 6°C. (IZ). Capillary counts
in organs of Ib did not differ from those found in
any of the corresponding ones in Ja and the re-
sults were pooled (J). In soleus and gastroc-
nemius, capillaries were counted only in densely
vascularized areas. There were 3 such areas in the
red fiber regions of the gastrocnemius which
covered 4% of the muscle in J and 7% in II, and
1 area in the soleus which covered 18% in J and
43% in© II. In the leg muscles, there were 85%
more capillaries/mm? in JJ than in J, except in
the white fibers of gastrocnemius where no change
was seen. There were also more smaller muscle
fibers/mm? in JZ, but, except for the soleus, the
ratio capillary/fiber was nevertheless higher in
II and in J (1.6 against 1.2). In liver and heart
there was no change in number of capillaries after
cold acclimation but in the ears there was a 12-
fold increase. In summary, cold acclimation had
the effect of increasing vascularization in ears
and leg muscles but not in liver and heart.

295. Measurement of velocity of blood by
means of ultrasound. J. F. Herrick, E. J.
Baupges, M. G. HauGEen* anp W. R. FarRRALL.*
Mayo Clinic, Rochester, Minn.

The use of ultrasound for measuring the velocity
of flowing liquids is not new. Several types of

Volume Ij

ultrasonic flowmeters have been described, some
of which have appeared in recent scientific jour.
nals. The work of Kalmus and his associates at
the Diamond Ordnance Fuze Laboratories and
the methods developed by Swengel have encour.
aged us to develop an ultrasonic flowmeter for
measuring the velocity of the blood. The method
will permit measurements on the unopened blood
vessel. Two barium titanate transducers, cylin.
drical in shape, have been applied to the outside
wall of plastic tubing or of excised blood vessels,
At one instant one of the transducers functions
as a transmitter of ultrasound and the other asa
receiver of the ultrasonic signal which has been
propagated through the moving blood. One
hundredth of a second later the functions of each
transducer are reversed. The electronic circuity
is designed to give information equivalent to the
difference between the time required to propagate
the ultrasonic signal upstream and the time for
propagation downstream (between the trans-
ducers). This information determines the velocity
of flow.

296. Effect of anti-malarial drugs on carbo-
hydrate metabolism of cardiac muscle
in vitro. Marityn E. Hess ann NreEts Hav-
GAARD (introduced by Grayson P. McCovca).
Lab. of Pharmacology, Univ. of Pennsylvania,
School of Medicine, Philadelphia.

The effect of quinine, quinidine and chloroquine
on the carbohydrate metabolism of rat heart
slices and homogenates has been studied. At a
concentration of 5 X 10-4 M quinine and quinidine
markedly depressed oxygen uptake and glucose
utilization of rat heart slices. A rat heart ho-
mogenate with a high KCl and DPN concentration
was also used. Added glucose is oxidized com-
pletely to carbon dioxide and water by this
preparation. The relatively slight depressant
effect of chloroquine upon heart slices could not
be attributed to lack of penetration of the drug
into the cells, since similar results were obtained
with the homogenates. In varying concentrations
quinine and quinidine each produced marked
inhibition of glucose utilization and oxygen up-
take by homogenates. Because quinine and quini-
dine affect cardiac carbohydrate metabolism to
greater extent than chloroquine, it is thought
either that the antifibrillatory action possessed
by these three drugs does not depend on their
effect on carbohydrate metabolism or that quinine
and quinidine have a different mechanism of
action from that of chloroquine.

297. Diuretic effect of nitrate salts. EpwiN
P. Hrarr. Dept. of Physiology, Univ. of North
Carolina School of Medicine, Chapel Hill.

The diuretic effect of single oral doses of NaNO:,

KNO; and NH,NO; was measured in dogs and

me
inj
ject

fibr

in 1

cell
tior
hep
tior
pro
fibr
cell

olume 1§

od, some
ific jour-
plates at
ries and
- encour-
.eter for
- method
ed blood
8, cylin-
> Outside
_ Vessels,
unctions
ther asa
nas been
rd. One
3 of each
circuity
it to the
ropagate
time for
> trans-
velocity

_ carbo-

muscle
Ls Hav-
Covucsa).
sylvania,

oroquine
vt, heart
d. At a
uinidine
glucose
eart ho-
ntration
ed com-
by this
pressant
ould not
che drug
obtained
trations
marked
gen up-
\d._ quini-
ism to &
thought
ossessed
on their
; quinine
nism of

, Epwin
of North
Ul.

'NaNO;,
logs and

March 1956

compared with the effect of NaCl, acetazoleamide
(Diamox) and intravenous meralluride (Mercu-
hydrin). These agents were given with 300 ml of a
sodium-poor vehicle (Lonalac) in doses of 7.5 mm/
kg for the salts, 500 mg for Diamox and 2 ml for
Mercuhydrin (78 mg Hg), to 3 trained dogs de-
prived of food and water for 24 hr. Estimates of
the net excretion of sodium, potassium, water
and milliosmols in the next 24 hr. were made by
subtracting the control excretion (vehicle alone)
plus the quantity administered from the total
amount excreted and expressed as u/kg/24 hr.
Net sodium excretion, probably the best diuretic
index, averaged around 3 mEq for Mercuhydrin,
Diamox and NH,NO;, 2 mEq for KNOs, 0.5 mEq
for NaNO; and —0.5 mEq for NaCl. Net volume
excreted was in approximately the same order,
averaging around 20 ml for Mercuhydrin, Diamox
and NH,NOs, 16 ml for KNOs;, 11 ml for NaNO;
and 3.4 ml for NaCl. Net potassium excretion
was about 2.8 mEq for NH,NO; and Diamox, 0.6
for NaNO; and negligible for the other agents.
Net osmolar excretion was negative for all the
salts presumably because of excretion of cation
with multivalent anions. Net osmolar excretion
after Diamox was 3.5 mOs and that after Mercu-
hydrin 1 mOs. No toxic effects were noted. (Sup-
ported by a grant from the American Heart Assoc.)

28. Fibroblastic participation in detoxifica-
tion of the amine, 48-80, by heparin. R. D.
HiGGINBOTHAM* AND Tuomas F. DouGHeErTy.
Dept. of Anatomy, Univ. of Utah College of Medi-
cine, Salt Lake City.

Mice pretreated (30 min.) with heparin, hyalu-
tonic acid or chondroitin sulfate, then challenged
with otherwise lethal doses of the phenylalkyl-
amine, 48-80 (6 ug/gm), were protected only by the
heparin. A straight line relationship between the
heparin 50% protective doses and their respective
48-80 challenge doses was obtained. Since heparin
and 48-80 form a precipitate in vitro, protection
may result from an in vivo formation of a complex.
Heparinized mice (500 ug, i.v.) challenged subcu-
taneously (100 ug 48-80, air pouch) had numerous
metachromatically granulated fibroblasts at the
injection site. A mixture of heparin and 48-80 in-
jected subcutaneously induced, within 30 min.,
marked metachromatic granulation in the local
fibroblasts, which closely resembled granulation
in mast cells. The majority of these granules ap-
peared to be digested within 24 hr., leaving the
tells with highly vacuolated cytoplasms. Substitu-
tion of chondroitin sulfate or hyaluronic acid for
heparin in this system induced little or no granula-
tion, suggesting a correlation between heparin
protection and the granulopoietic response. The
fibroblastic ingestion of macromolecular or mi-
cellar polysaccharide structures has been desig-

AMuRICAN PHYSIOLOGICAL SOCIETY 93

nated as ‘micellophagosis’ (Hice1nBoTHaM, R. D.
et al., Federation Proc. 14 (1): 232, 1955). This
process may function as a normal mechanism in
the maintenance of the connective tissue, being
adaptable to detoxification relative to the nature
of the polysaccharides (as well as of the noxious
agents) in the ground substance. It is interesting
that the metachromatic granules of mast cells are
thought to contain both heparin and histamine,
as well as 5 hydroxytryptamine (Brennirt, E. P.,
Proc. Soc. Exper. Biol & Med. 90: 303, 1955). (Sup-
ported by Grant #DA-49-007-MD-130, Dept. of
the Army.)

299. Metabolism of premature and full-term
infant brain. W1LLIAMINA A. Himwicu, H. B.
W. Benaron* AND Beatrice E. Tucker.* Gales-
burg State Research Hosp., Galesburg, and North-
western Med. School, Chicago, Iil.

The oxygen consumption of various parts of the
brains obtained from premature and full-term in-
fants have been measured using glucose as a sub-
strate and potassium phosphate at pH 7.4 as buffer.
The brains were removed as soon as possible after
death. The infants ranged in gestation age from
23 to 40 wk. The parts studied were the frontal cor-
tex, caudate nucleus, thalamus, superior colliculi,
medulla oblongata and cervical spinal cord. Great
care was taken to sample each time the same area
of the part being studied. Data obtained from 4
stillborn infants who were otherwise in good con-
dition at the time of delivery are included since
their inclusion does not change the averages for
the group as a whole. Although the highest oxygen
consumption was obtained from the brain of a full-
term infant, the increase over values obtained
from a 23 wk. premature was not great. In approxi-
mately 4 of the brains the medulla showed the
highest oxygen consumption of all the parts stud-
ied; in 3 the frontal cortex was highest. The cau-
date nucleus in most cases has the lowest level of
metabolism judged by the oxygen uptake. The
possible physiological and functional significance
of these data will be discussed. Since these data
are of fundamental importance in the field of ob-
stetrics, the results will be published in extenso in
the literature of that field.

300. Inhibition of yeast alcohol dehydrogen-
ase by metal-binding agents. FrRreprEric L.
Hocu anp Bert L. VALLEE (introduced by
CHARLES P. Lyman). Biophysics Research Lab.,
Dept. of Medicine, Harvard Med. School and Peter
Bent Brigham Hosp., Boston, Mass.

Yeast alcohol dehydrogenase (ADH), contain-
ing 4 functional atoms of zine per molecule, has
previously been shown to be inhibited by certain
agents having high affinity for zinc (VALLEE, B. L.
AND F. L. Hocu. Proc. Natl. Acad. Sci. 41: 327,

94 FEDERATION PROCEEDINGS

1955). Yeast ADH is now shown to be inhibited
by other metal-binding agents such as BAL, thio-
semicarbazide, semicarbazide, azide, cupferron
and diethyldithiocarbamate. The degree and rate
of inhibition are a function of the concentration of
inhibitor and the pu, time and temperature of in-
cubation of the agents with ADH. Activity is not
inhibited by ethylenediamine tetraacetic acid, ni-
trilotriacetic acid, triethylenetetramine, ethylene-
diamine and imidazole. These findings will be dis-
cussed with reference to the known characteristics
of the complexes of zinc with these agents in solu-
tion; the mixed complexes of [(ADH)Zn,] with
chelating agents will also be discussed. The effects
of DPN, DPNH, ethanol, and acetaldehyde on the
inhibition produced by incubation with ortho-
phenanthroline (OP) have been studied in detail.
The degree of inhibition is reduced only when
DPN is added to ADH at the same time or before
OP is added. When DPNH, ethanol or acetalde-
hyde are added to ADH before OP, inhibition is
more rapid and complete than when OP is added
alone. DPN competes with OP, but DPNH does
not, as shown by kinetic experiments. It thus ap-
pears that the zine of [(ADH)Zn,] is a site of inter-
action between DPN and ADH, an interaction
which differs from that of DPNH with [(ADH)Zn,]
under these conditions.

301. Body temperature variations of nonhi-
bernating Alaskan ground squirrels. Ray-
MOND J. Hock. Arctic Aeromedical Lab., Ladd
Air Force Base, Fairbanks, Alaska.

It is well known that hibernating mammals
when active have more labile body temperatures
than do true homoiotherms. This study reports
observations on 77 Alaskan ground squirrels
throughout four seasons 1951-54 from April 23 to
Oct. 10, the entire period of activity. The animals
were shot in the wild, and deep colonic tempera-
ture taken immediately. Mean for the entire pe-
riod wag39.0°C (extremes 33.0°-41.0°). Thirty ani-
mals captured at the same time and killed after a
few days of captivity had mean temperature of
37.8°C (34.0°-39.0°), and closely paralleled wild-
shot animals. Long term captive animals at all
times of year, including active season, exhibited
lower mean temperature and larger extremes of
range. Mean of 92 squirrels in summer season was
34.9°C (30.0°-38.0°); mean of 62 active squirrels in
winter season was 34.6°C (30.0°-39.0°) (below
30.0°C considered to be hibernating or in transi-
tory state). No parallelism was shown between
captive and wild squirrels. No correlation was
found in wild series between body temperature
and weight, sex, ambient temperature or events
in the breeding cycle. There thus seem to be 4
phases in the annual body temperature cycle of
ground squirrels: /) the low temperature of hiber-

Volume 1§

nation, continuing from early October to about
April 20 (poikilothermism) ; 2) a variable tempera-
ture from emergence until late June (heterotherm.
ism); 3) a period of relatively constant tempera-
ture, from late June through early September
(homoiothermism); 4) a period of lowering tem.
perature in late September and early October, pre-
ceding hibernation (heterothermism).

302. Graded response in the dog heart muscle,
B. F. Horrman anp C, Y. Kao.* Dept. of Physi-
ology and Pharmacology, State Univ. of New York,
College of Medicine at New York City, Brooklyn.
Graded responses are prominent manifestations

of electrical activity of the neuromyal junction,
synapses and some invertebrate muscle fibers. In
nerves (Kao AND GrunpFEsT, Biol. Bull. 1955)
and skeletal muscle fiber (EccLEs AND O’Connrr,
J. Physiol. 1941) they are revealed only when the
normally superseding all-or-none responses are
inoperative during refractoriness or drug action.
Graded responses in dog Purkinje fibers have been
recorded by means of intracellular microelectrodes
from a preparation consisting of a papillary muscle
and attached bundles of Purkinje fibers. Action
potentials of papillary muscles, lasting only half
as long as those of Purkinje fibers, are used to
initiate impulses successively in the latter tissue
during the repolarization phase. When re-excita-
tion was induced early in the refractory period,
transmembrane recording from a Purkinje fiber
close to its junction with papillary muscle revealed
non-propagated decremental responses occasion-
ally having reversed membrane polarity. Stimula-
tion later during repolarization elicited normally
propagated all-or-none responses in distant por-
tions of the fiber. These originated from activity
in more proximal areas where the propagated ac-
tion potential arose out of a local response. The
absolute refractory period thus determined corre-
sponded approximately to that obtained by ap-
plied cathodal stimuli and demonstrates that the
stimulating efficacy of the cardiac action potential
is considerably greater than the threshold require-
ment.

303. Effect of temperature on whole blood
buffers. D. A. Hotapay, E. NELSon Anp V.
LAUDERDALE (introduced by Tuomas H. AL-
LEN). Dept. of Anesthesiology, Columbia Univ.,
New York City.

Evaluation of both the respiratory and meta-
bolic components of acid-base balance requires
determination of blood pu, COz content and hema-
tocrit, and a knowledge of the buffering charac-
teristics of the nonbicarbonate anions of whole
blood. These characteristics are known for blood
proteins at 37°C (Stncer AnD Hastinas, Medicine
47: 223, 1948) but not at other temperatures. The

Se: tS

olume 15

0 about
empera-
otherm-
empera-
ptember
ng tem-
ber, pre-

muscle,
of Physi-
ew York,
rooklyn,
Stations
unction,
bers. In
ll. 1955)
JONNER,
rhen the
ses are
+ action.
uve been
ectrodes
y muscle
. Action
nly half
used to
ar tissue
+-excita-
period,
ije fiber
revealed
ccasion-
Stimula-
ormally
ant por-
activity
ated ac-
ise. The
d corre-

by ap-
that the
otential
require-

» blood
AND V.
H. At-
r Univ.,

d meta-
requires
d hema-
charac-
f whole
or blood
Uf edicine
res. The

March 1956

effect of change of temperature on the dissociation
of the nonbicarbonate buffers of whole blood was
determined by comparison of the CO: combining
power of fully oxygenated, heparinized human
blood at 2 different temperatures. A portion of
each sample was equilibrated with an O.-CO: gas
mixture in a tonometer at 37°C, and a 2nd portion
at another temperature between 21° and 37°C.
pH was measured at the equilibration temperature
with a glass electrode and hematocrit, O2 capacity,
whole blood O, and CO: contents, and plasma CO:
content were determined immediately. Whole
blood bicarbonate content of each portion was
calculated. Buffer base of the 37° portion was esti-
mated from the nomogram of Singer and Hastings.
The anionic value of the nonbicarbonate buffers
was obtained by subtraction of the whole blood
bicarbonate from the buffer base (which is the
same in both portions). It was observed in 35 com-
parisons on samples from 32 subjects that the non-
bicarbonate buffers decreased an average of 0.672
mEq/l/degree reduction of temperature (S.D.
0.141 mEq/l.; S.E. 0.024 mEq/I.). No significant
correlation with hematocrit or pH was observed.

$4. Causes of basal secretion of HCl in the

dog. FRANKLIN HOLLANDER AND VERNON A.

WEINSTEIN.* Gastroenterology Research Lab.,

Mount Sinai Hosp., New York City.

Basal or ‘spontaneous’ gastric secretion arises
from a multiplicity of stimuli. The importance of
the roles of vagal and postprandial secretagogue
activities are well recognized. In order to study
other possible causes, subcutaneous Heidenhain
pouches were prepared with the omentovascular
pedicle passing through a small opening in the
abdominal musculature and peritoneum. Such ani-
mals frequently gave evidence of basal HCl secre-
tion even 2 and 8 days after the last meal. Preven-
tion of coprophagy and skin licking failed to
reduce the incidence of this occurrence. In order
to test the possible role of sympathetic secretory
fibers, the pedicle was transected intraperitoneally
after development of a collateral circulation. This
conversion of the pouch to a Bickle-type also
failed to eliminate residual basal activity. Finally,
to investigate the possible occurrence of gastrin
activity unrelated to eating, an antrectomy (with
gastroenterostomy) was performed. This opera-
tion eliminated the remainder of basal secretion in
all surviving dogs. These animals and several
others similarly prepared have been in use for1 yr.
or more. Gastric pH, observed for 1 or more hr.
after a 24-hr. fast, has invariably been 7 or higher
except on rare occasions when it was between 6
and 7. Conclusion: in addition to cholinergic and
dietary secretagogue mechanisms by which basal
secretion is evoked in the dog, there is also a
modicum of gastric hormonal stimulation; spon-

AMERICAN PHYSIOLOGICAL SOCIETY 95

taneous activity of parietal cells does not occur.
(Aided by Grant C 288 National Cancer Inst.,
Natl. Insts. of Health, PHS.)

305. Purification of the lipemia clearing fac-
tor of postheparin plasma. CHARLOTTE HoL-
LETT* anD H. C. Mena. Dept. of Physiology,
Vanderbilt Univ. Med. School, Nashville, Tenn.
A procedure for the purification of the lipemia

clearing factor of postheparin plasma has been de-

veloped. It was possible to obtain a purified clear-
ing factor with a 1480-fold increase in specific
activity over that of postheparin plasma. The pro-
cedure includes precipitation of postheparin
plasma, diluted 1:15 with water, at px 5.7. The
resulting precipitate (Fraction I), was dissolved
in 0.06 m phosphate buffer, pH 7.8. The subsequent
procedure, performed at 0°C, was as follows: Frac-
tion I was brought to 25% saturation with am-
monium sulfate (saturated at 0°C, px 5.70). After
centrifugation, the supernatants were brought suc-
cessively to 33%, 40%, 50%, and 64% saturation
with ammonium sulfate. The precipitates at each
step were stored at —20°C. The various precipi-
tates were dissolved in 0.06 m phosphate buffer and
dialyzed at 4°C against phosphate buffer and as-
sayed for clearing activity and protein content.

The precipitate obtained at 64% saturation had a

specific activity of 36 u/mg (cf. postheparin

plasma, 0.025 u/mg). (One unit is the amount of

clearing factor which produced a decrease of 0.100

in optical density in 1 hr.) This gave an increase

of 1480 in specific activity, with a yield of 37%.

Properties of the purified clearing factor include:

1) optimum pu 8.50; 2) stable for 1 wk. at 4°C; 3)

heat labile; 4) albumin or nonheparinized plasma

is required for clearing to occur; 5) Fraction III-0

of plasma proteins cannot replace albumin; 6)

inhibited by sodium taurocholate (4 X 107! m);

7) calcium chloride had no effect on clearing ac-

tivity; 8) the u.v. absorption peak is 279 mu.

306. Simultaneous and independent deter-
mination of right ventricular and left ven-
tricular end-diastolic (EDV), end-systolic
(ESV) and stroke volumes. J. P. Hout, J. W.
ALLENSWoRTH,* D. M. Cotuins* AND M. Fine.*
Inst. for Med. Research, Univ. of Louisville,
Louisville, Ky.

In these studies the EDV, ESV and Stroke Vol-
ume were measured simultaneously and independ-
ently in the anesthetized dog by means of the elec-
trical conductivity technique (Hout, J. P. anp
J. Wimpy. Federation Proc. 14: 484, 1955). The
EDV of the left ventricle had no consistent rela-
tionship to the simultaneous EDV of the right
ventricle; at one time during an experiment the
left ventricular EDV would be larger and a short
time later it would be smaller than the simul-

96 FEDERATION PROCEEDINGS

taneously determined right ventricular EDV. Sel-
dom did the 2 ventricles have the same EDV. In
some experiments, the EDV of one ventricle was
always greater than the EDV of the other ven-
tricle, but the difference between the two varied
from time to time. Similar results were obtained
for ESV and Stroke Volume. The average of a
number of determinations of the left ventricular
Stroke Volume was approximately the same as
that of the right ventricle. Both ventricles always
empty in a ‘fractional’ manner and preliminary
observations suggest that with any stroke the 2
ventricles empty by the same fraction, that is if
the left ventricle ejects 48% of its EDV then the
right ventricle simultaneously ejects 48% of its
EDV, although the simultaneous end-diastolic
volumes of the 2 ventricles differ. (Supported in
part by Grant No. 2075, National Heart Inst.,
PHS, the American Heart Assoc. and the Ken-
tucky and Louisville Heart Assocs.)

307. Effect of arterial pressure level on site of
development of arteriolar necrosis in dogs
malignant hypertension. Ernest L. Hop-
Kins,* RoBERT S. JASon* AND Epwarp W. Haw-
THORNE. Depts. of Physiology and Pathology,
Howard Univ., College of Medicine, Washington,
D.C.

This study was undertaken to assess the role of
the arterial pressure level in the pathogenesis of
the vascular changes accompanying experimental
malignant hypertension. In our experience the
ileum of the malignant hypertensive dog shows
extensive macroscopic, and microscopic evidence
of vascular damage in the form of hemorrhage and
necrosis. This group of experiments was designed
to determine whether isolated arterial pressure
reduction in a segregated portion of ileum would
modify the development of necrosis in the arte-
rioles of the low pressure segment while at the
same time permit the necrosis and hemorrhage to
occur in¢he adjacent ileum of dogs succumbing to
experimental malignant hypertension. Malignant
hypertension was produced in this series of ani-
mals by severe simultaneous bilateral renal artery
constriction (BRAC). Prior to this procedure and
at the same operation an isolated segment of ileum
under low pressure was constructed by constrict-
ing one of the intestinal branches of the mesenteric
artery with a ‘monkey kidney clamp’ until the
visible as well as the palpable pulsations were
approximately 3:1, the arcuate branches of this
vessel were ligated at their distal ends to prevent
collateral inflow from the sub-mesenteric anasto-
moses and an isolated segment of ileum was thus
obtained. In such animals malignant hypertension
and death occurred in an average of 72 hr. follow-
ing BRAC. In each of 6 animals successfully pre-
pared we observed both macroscopic as well as

Volume 16

microscopic differences in the two segments of
ileum. The segment under low pressure showed no
evidence of arteriolarnecrosis.

308. Reactions of nude men to a mild cold ex.
posure. Steven M. Horvatu, G. B. Spurr,*
B. K. Hurt* anp L. H. Hamixton.* Dept. of
Physiology and Cardiovascular Lab., State Univ,
of Iowa, Iowa City.

Ten nude male subjects were exposed a total of
15 times for a 12-hr. period to an environment hay-
ing an ambient temperature of 15°C and a relative
humidity of 35%. The subjects were first main-
tained for a 24-hr. period in an environment of
24°C and 50% relative humidity and were returned
to this environment for further tests following
their cold exposure. Body temperature, metabo-
lism and some cardiovascular reactions were meas-
ured before and after 1, 5 and 10 hr. of cold expo-
sure. The rectal temperature did not change as a
consequence of the cold exposure. However, be-
cause of the fall in mean skin temperature, the
mean body temperature decreased from a control
value of 35.2° to 33.5°C. Heat production increased
from a control of 41.3 Cal/m?/hr. to a peak value
of 63.9 Cal/m?/hr. during the 5th hr. in the cold,

309. Renoprival hypertension in totally sym-
pathectomized dogs. C. R. Houcx!. Div. of
Physiology, Univ. of Tennessee Med. Units,
Memphis.

Eight dogs were totally sympathectomized in
4-stage operations separated by at least 1 wk. The
animals were allowed a recovery period of 1 month
and were then bilaterally nephrectomized in one
stage. Thereafter the dogs were maintained as far
as excretory function and normal hydration is con-
cerned for 10-14 days by intermittent peritoneal
dialysis. The dialyses were performed 3 times a
day for the 1st postnephrectomy week and twice
a day thereafter. All of the animals developed hy-
pertension (at least 30 mm Hg) before the 7th day
postnephrectomy and in this regard do not differ
from nephrectomized nonsympathectomized dogs.
Three animals had blood pressures of more than
80 mm Hg above control at some time during the
renoprival period and all sustained increments of
at least 44 mm Hg during the study. With a single
exception, the hypertensive levels were main-
tained throughout the postnephrectomy period.
In 1 dog which became dehydrated by the 9th day,
the blood pressure fell to a level 10 mm Hg below
control pressure only to rise to hypertensive levels
again upon rehydration. These data point to the
dispensability of the sympathetic nervous system
in the etiology of renoprival hypertension. (Sup-
ported by a grant-in-aid from the American Heart
Assoc.)

1 Deceased.

olume 16

nents of
owed no

cold ex.
Spurr,*
Dept. of
te Univ,

total of
ent hay-
relative
3 main-
ment of
returned
ollowing
metabo-
re meas-
ld expo-
nge asa
ver, be-
ure, the
control
icreased
ik value
he cold,

ly sym-
Div. of
Units,

1ized in
wk. The
1 month
1 in one
d as far
1 is con-
ritoneal
times a
id twice
ped hy-
7th day
t differ
1d. dogs.
re than
‘ing the
.ents of
a single
. main-
period.
th day,
s below
e levels
, to the
system
. (Sup-
1 Heart

March 1956

0. Biological effects of dehydroepiandros-
terone. EvEtyN Howarp. Johns Hopkins Med.
School, Baltimore, Md.

Dehydroepiandrosterone (DHA), a weak andro-
go in capon comb tests, has been shown by
Migeon to be the chief 17-ketosteroid identifiable
in human plasma and is presumably of adrenal
gigin. In doses of 0.14 mg/day it produces no en-
jrgement of the seminal vesicles in castrated
nice, but does suppress the adrenal X zone, with a
0% reduction in adrenal weight. Corticosterone
B) reduces the width of the zona fasciculata rela-
ive to X, and gives a 29% adrenal weight reduc-
tion at a dose of 0.4 mg/day. DHA and B adminis-
ered simultaneously produce an extremely narrow
ortex and an adrenal weight reduction of 69%.
Histological findings will be discussed. The ob-
grvations support the view that there are 2 sepa-
mte trophic factors influencing the adrenal. Preg-
senolone did not reduce adrenal weight. Although
inactive in the seminal vesicle test, DHA causes
wnsiderable enlargement of the female preputial
gands and of the phallus in gonadectomized mice
either sex. There is a particularly striking en-
largement of the clitoris in adrenalectomized
wariectomized infantile mice on DHA pellets.
DHA has little effect on the uterus in ovariecto-
nized adrenalectomized mice, but it does promote
gowth of the uterus in adrenalectomized mice
(n doca) and it tends to overcome the anti-
mabolic effects of corticosterone with respect to
werine growth in the presence of the ovaries.
The findings are consistent with the view that
DHA may be of significance as an antagonist to
orticoids during growth of some tissues. (Aided
by PHS Grant H 2414.)

il. Relationship of oxygen debt to blood
lactate and pyruvate. WILLIAM E, HucKABEE*
anD JuLius SenpRoy, JR. Div. of Chemistry,
Naval Med. Research Inst., Bethesda, Md.

In experiments on dogs under light chloralose
mesthesia, there were obtained 31 O2-debt esti-
nates from 5 dogs subjected to various intensities
ifexercise by electrical stimulation, and 90 meas-

ements from 18 dogs under conditions of respira-
fry hypoxia (breathing 8-15% O2). The results
thowed that blood lactate levels in recovery did
wt serve to indicate the extent of development of
(debt; hence, these values merely obscure the
netabolic basis of debt. Measurements of arterial
ind mixed-venous blood oxygen and of cardiac
utput likewise failed in serving to estimate the
degree of generalized hypoxia in the same sense
8 oxygen debt. Present concepts of tissue energy
jroduction suggest that total lactate production
i hypoxia can be corrected for the mass action
lect of pyruvate changes if these are measured,
having a moiety, ‘excess lactate’, which alone

AMERICAN PHYSIOLOGICAL SOCIETY 97

should be related to oxygen debt development.
Oxygen debt values, based on the oxygen-equiva-
lent of lactate distributed throughout the total
body water, were calculated from total, and ‘ex-
cess’, blood lactate levels. The data so obtained
showed a wide divergence in all groups, between
respiratory debt and total blood lactate. However,
there was a very close correlation, within the
limits of analytical error, between ‘excess’ lactate
production (debt), and oxygen debt. These experi-
ments suggest 1) that blood total lactate concen-
tration is unrelated to body oxygen debt, 2) that
the rate of ‘excess lactate’ production, a simple
function of changes in both pyruvate and lactate,
has a high predictive value for body oxygen debt,
and 3) that oxygen debt estimation gives a unique
measure of tissue metabolic oxygen deficiency.

312. Action of metabolic inhibitors and drugs
on isolated frog skin. Ernst G. Hur. Dept.
of Physiology, Med. College of Virginia, Rich-
mond.

Normal frog skin contains 350 nEqNa*/gm and
180 wEqKt/gm of dry skin. Such skins actively
transport NaCl from the outside to the inside of
the skin. Skins in 2 X 10-* molar azide-salt solu-
tion (115 nhEqNa*, 10uEqKt/ml) maintained nor-
mal Nat and K* content, but the rate of active
NaCl transport dropped to about } of the control
value. Qo. was reduced by 25% of the control Qoz.
Skins in 1 X 10-* molar azide solution increased
their Na* and decreased their K+ content (500
pEqNa*/gm; 125 hEqK*/gm); active NaCl trans-
port was completely inhibited; Qo, decreased 53%.
Similarly for cyanide and 2-4 DNP, concentra-
tions could be found that drastically inhibited
active NaCl transport, leaving electrolyte content
of the skin intact. Monoiodoacetate, however, in-
hibited active transport only at doses that would
also lead to increase in Nat and decrease in Kt
content of skin. Increase in active transport asso-
ciated with maintenance of Nat and K+ content
of skin was seen in the presence of theophylline
and, to a smaller degree, of Mersalyl. Qo. was
moderately increased. CO (90% + 10% Oz) had no
effect on Na*-K*t content and active Na* trans-
port. The results suggest that maintenance of nor-
mal Na* and Kt content and active NaCl trans-
port across the skin are independent cell functions
that may be separated from each other by meta-
bolic inhibitors acting on cytochrome oxidase and
mechanisms of oxidative phosphorylation. It is
visualized that the two cell functions have differ-
ent locations within the epithelial cells or differ
in their chemical characteristics, or differ in both
respects. (Supported by research grant RG-3545
(C 3), Natl. Insts. of Health.)

313. Types of posttetanic potentiation in au-
ditory and visual systems. JoHN R. HucHeEs

98 FEDERATION PROCEEDINGS

(introduced by I. Tasaxr). Natl. Inst. of Mental

Health, Natl. Insts. of Health, Bethesda, Md.

The increased responsiveness in the nervous
system following repetitive stimulation has been
termed posttetanic potentiation (PTP). Both psy-
chophysical and electrophysiological experiments
have shown types of PTP in the auditory system.
In the psychophysical study on humans, monaural
thresholds were determined continuously with a
recording audiometer before and after the ear was
exposed to a low tone. After the exposure there is
a brief rise in threshold, a lowering and then a
second rise. This increased sensitivity (potentia-
tion) lasts for approximately 2 min. after the ces-
sation of the exposure tone. The electrophysio-
logical study was conducted to determine the
effect of low-tone exposure on the amplitude of the
neural response recorded from the round window
of cats. After the exposure, the first neural re-
sponse (N;) of a test tone shows a brief subnor-
mality, a sensitization (potentiation) and then a
second subnormality. This potentiation lasts for
approximately 2 min. after the cessation of the
exposure tone. Electrophysiological evidence has
been found for the existence of PTP in the visual
system. The cat’s optic nerve was stimulated and
potentials were recorded in the lateral geniculate
and on the visual cortex. A brief tetanus was ap-
plied during a tetanically induced subnormal
phase. Compared with the pretetanic level, the
response after the tetanus showed a brief subnor-
mality, a long-lasting potentiation and then a
second subnormality. The potentiation lasts up to
7 min. Similarities in these 3 types of PTP suggest
that the processes underlying the psychophysical
and electrophysiological data may possibly be
related.

314. Effects of various respiratory impedances
on pulmonary ventilation and alveolar gas
exchange in anesthetized dogs. W. E. Hutt
ann ®.G. Hau. Dept. of Physiology and Pharma-
cology, Duke Univ. School of Medicine, Durham,
N.C.

Respiratory obstruction was produced in anes-
thetized dogs by insertion of plate orifices of vari-
ous diameters into tracheal air streams. Imped-
ance to breathing was correlated with breathing
patterns, pulmonary ventilation, arterial blood
pressure, arterial oxygen saturation and arterial
carbon dioxide tensions. It was found that as im-
pedances were increased, tidal volumes became
more effective in accomplishing alveolar ventila-
tion as measured by arterial oxygen saturation.
These experiments also give a measure of the
potency of respiratory reflexes in meeting the de-
mands for respiratory gas exchange.

315. Effects of chloretone and of azide on res-
piration and ionic content of frog nerves.

Volume 15

W. P. Hurisut,* Tomoakt AsANo* Anp fF

Brink. Rockefeller Inst., New York City.

Sodium azide (p.2-5 mm) and chloretone (1-5
mm) depress the rate of oxygen uptake of resting
nerves. Two millimolar chloretone or 0.2 mM azide
lowers this rate about 20% without measurable
change in the sodium or potassium content of the
nerves. Higher concentrations of these reagents
cause loss of potassium and gain of sodium. The
lowered respiratory rates are established quickly
but the ionic changes continue with decreasing
rates for more than 10 hr. For azide that ionic
changes, developed in 5 hr., are correlated with }1.
the degree of depression of the respiratory rate. A ] ec
similar correlation is obtained for chloretone (2-5 | M
mm). Fifteen millimolar chloretone produces a § 7
further marked increase in leakage of ions without } A
a correspondingly large increase in respiratory de- fie
pression. Azide may affect the ionic distribution Jism
in nerves solely by inhibition of oxidative proe- fidi
esses while chloretone, a narcotic, may in addition }m
act directly upon the fiber surfaces. One hour of },¢/1
activity at 50 volleys/sec. in A-fibers also produces §uir
loss of potassium and gain of sodium. The magni- fis
tudes of the ionic changes decrease as the potas- },vel
sium concentration of the bathing solution is in-
creased, being just detectable in 8.5 mm potassium,

tite
Piast
cate
cont
fi
thio
rac

had been removed.

316. Rates of cooling of rats in the cold. P. Ff,
IampretTrRO, E. R. Buskirk AND M. J. FREGLY

search and Development Command, Natick, and
Harvard Med. School, Boston, Mass.

nance of body temperature of rats immobilized fiy,
and exposed to cold air (5°C) it became necessary Fits

various treatments (adrenalectomy, propylthio-
uracil administration (in food), death) on CCR.
Colonic temperatures were recorded continuously
by means of a recording potentiometer. All rats
were cooled until colonic temperature fell to
22.5°C. In normal animals a relationship between § h
body weight and CCR was found which is best B
described by the equation: log CCR = 2.776 - E
0.886 log body weight. In view of this relationship, H
animals in experiments described below were se-
lected in the weight range 200-260 gm. In this hy
limited range CCR could easily be corrected for

the effect of body weight from a linear portion of ,
the above curve as follows: CCR = 7.51 — 0.0
body weight (+0.004 S.E.) Adrenalectomized rats om

cooled 60% faster than sham-operated controls.

Yarch 1956

Volume 15

popylthiouracil-treated rats cooled 35% faster.
When rats were both adrenalectomized and treated
yith propylthiouracil CCR was increased 77%.
the effects of adrenalectomy and propylthiouracil
ministration on CCR do not appear to be quan-
jitatively additive. Dead animals cooled 152%
qster than controls. In summary, the results indi-
ate a family of cooling curves ranging between
witrols and dead animals in the following order
increasing rates of cooling: controls, propyl-
hiouracil-treated, adrenalectomized, propylthio-
yacil-treated plus adrenalectomized, dead.

*
Ly.
tone (I-15
of resting
MM azide
1easurable
ent of the
> Teagents
lium. The
d quickly
lecreasing
that ionic
ated with
ry rate. A
etone (2-5
roduces a
1s without
ratory de-
stribution
tive proe-
1 addition
e hour of
) produces
he magni-
he potas-
ion is in-
otassium,
by 0.2 mu
the extra
periments
rineurium

AND Ff,

7. Alterations in iodide metabolism during
cold exposure. ALFRED InToccIA* AND L. VAN
MippLEeswortuH. Dept. of Physiology, Univ. of
Tennessee, Memphis.

Aseries of experiments were undertaken to de-

mine the effects of cold on the iodide metabo-
jgm of rats. Six rats were fed 10 gm/day of a low
idide goitrogenic diet (thyroid weights 38 mg/100

mb. wt.). This diet was tagged with I’ (0.14
g/rat at beginning of experiment) daily and the
uimals were allowed to reach equilibrium with
his diet. At equilibrium the fecal I!*! excretion
weraged 60% of the daily I'*! dose and the urinary
averaged 35%. Radioactivity determined over
he thyroid area before equilibrium revealed a pro-
gessive increase in thyroidal I!*! accumulation.
When equilibrium was attained the percentage of
the daily I'#! accumulated by the thyroid no longer
increased but remained at 345%. After equilibrium
man I'*! tagged diet at 27°C, the rats were ex-
psed to 13°C. No appreciable change was noted
nfecal and urinary I[}*! excretion after 1 wk. at
}°C. Lowering the temperature to 10°C produced,
iter 1 day, a reversal in the fecal and urinary ex-
metion pattern. The urinary I!*! increased to 70%
ithe daily intake and fecal I'*! decreased to 30%.
Mfter 2 days at 10°C, the radioactivity over the
yroid area, expressed as percentage of the daily
take, increased from 345% to 452% (P = 0.02).

These data may indicate that one of the first meta-
lic alterations in cold is an increased deiodina-

ion of thyroid hormone, resulting in more endoge-

wus iodide available to the thyroid and kidneys.

Id. P.F.
. Free
raster Re-
utick, and

e mainte-
mobilized
necessary
eight and
effect of
opylthio-
on CCR.
tinuously
. All rats
» fell to
. between
h is best
2.776 -
tionship,
were se-
. In this
ected for
ortion of
1 — 0.01
ized rats
controls.

8. Metabolic effects of intravenously ad-
ministered triiodothyronine in euthyroid
human subjects. Marian C. Isaacs,* HELEN
B. Horow1tz,* Bernarp A. Sacus,* ALVIN
Essia* AND RaymMonp E. Weston. Montefiore
Hosp., New York City.
In 2 euthyroid subjects and 1 labile mild hyper-
hyroid patient whose basal control oxygen con-
ption and radioiodine uptake were normal,
ily metabolic balances for Na, Cl, K, N, P, Ca
d H.O; daily urinary exeretion of creatinine,
atine, sulfur, total solutes and 17-OH-corticoids;
rresponding blood chemistries, total proteins

AMERICAN PHYSIOLOGICAL SOCIETY 99

and lipids and oxygen consumptions were deter-
mined before, during and after daily intravenous
injections of triiodothyronine (0.4-1.8 mg) for 4-6
days. Headache, emotional lability and tachy-
cardia promptly developed as did signs of in-
creased catabolism, including decreased blood
lipids, increased urinary total solute and creatine
excretion and initially negative potassium, nitro-
gen and disproportionately greater phosphorous
balances. Subsequently, urinary potassium de-
creased but urinary nitrogen, like the oxygen con-
sumption which rose 50 to 80%, continued to
increase and in 1 subject reached 20 gm/day. Cal-
cium excretion increased in 1 normal subject but
not in the other whose control excretion rate was
very low. No striking changes in sulfate or 17-OH-
corticoids occurred. On the 2nd and 3rd experi-
mental days, all patients, including the 2 on low
sodium intakes, exhibited significant natriuresis
and chloriuresis, followed by increased salt reten-
tion until 2-3 wk. later when natriuresis and chlor-
iuresis recurred for 4-5 days. After triiodothyro-
nine was discontinued, clinical and laboratory
evidence of hypermetabolism persisted for 4-6
days. Then, as oxygen consumption fell, balances
became positive. However, in the euthyroid sub-
jects the total body weight and nitrogen pre-
viously lost were not regained even after 26-41
days on the 2000-2400 cal. metabolic diets.

319. Sodium and potassium exchanges across
isolated thymus nuclei. S. Iton* anp I. L.
Scuwartz. The Rockefeller Inst. for Med. Re-
search, New York City.

In experiments on thymus nuclei isolated in
sucrose it was found that Nat was necessary for
C14-amino acid to be incorporated into proteins of
the nucleus (ALLFREY, Mirsky aNnp Osawa, Na-
ture 176: 1042,.1955). Nuclei, prepared by Dr. All-
frey in 0.25: mM. sucrose containing 0.002-0.003 m
CaCl, contained 75+4.9 wEq of sodium and 261+
26.2 wEq of potassium/gm d. wt. When nuclei were
incubated at 37°C for 60 min. in sucrose-phosphate
buffer (pH 6.2; K 73 wEq/ml; Na 33 wEq/ml) the
content of sodium and potassium increased over
preincubation values by 78 and 97%, respectively;
in contrast, at 0°C the content of sodium and po-
tassium increased over initial values only by 45
and 8.3%, respectively. These findings were not
altered by adding glucose to the medium. In a
potassium-free sucrose-phosphate buffer (px 6.2;
Na 77 wEq/ml), the nuclei lost 50% of their origi-
nal potassium content on standing for 60 min. at
0°C, but lost only 19% on incubation for 60 min. at
37°C. The nuclei gained an average of 85 and 55
nEq of sodium/gm d. wt. at 37°C and 0°C, respec-
tively. Hg++, Cu++, Bat+, Mgt* and Ca*+ (Catt
increased to 0.005 to 0.03 m) inhibited accumula-
tion of potassium and sodium from the high potas-

100

sium medium. 2:4-dinitrophenol (2 X 10~‘ m), so-
dium azide (10-* m), and potassium cyanide (10-*
a) partially inhibited potassium uptake by the
nuclei from the high-potassium medium and facili-
tated potassium loss and sodium gain from the
potassium-free medium.

320. Temperature effects on the swelling of
liver slices immersed in solutions of sodium
chloride, monosaccharides and disaccha-
rides. 8. Iron* ann I. L. Scuwartz. The Rocke-
feller Inst. for Med. Research, New York City.
Slices of liver taken from rats immediately after

death and immersed for 10 min. in various fluids

appear to be isotonic with 0.35 m NaCl at 4°C and
20°C, with 0.25 m NaCl at 37°C, with 0.65 m glu-
cose at 20°C and 37°C, with 0.46 m glucose at 4°C
and with 0.34 m sucrose at 4°C, 20°C and 37°C.

Similar results were obtained when glucose was

replaced by fructose or mannitol and when sucrose

was replaced by lactose and maltose. The inulin
space of slices maintained at 0°C did not differ
significantly from the inulin space of slices main-

tained at 20°C or 37°C. When liver slices were im-

mersed for 1-60 min. in 0.3 m NaCl the entry of

water, chloride and Na + K was significantly and
without exception less at 37°C than at 20°C; but
in 0.66 m glucose the entry of water and presuma-
bly glucose was significantly and without excep-
tion less at 20°C than at 37°C. Therefore, the
greater swelling of liver slices immersed in sodium
chloride solutions at 4°C and 20°C, as compared
with slices immersed at 37°C, appears to be due at
least in part to failure of solute (sodium and chlo-
ride) extrusion at the lower temperatures. In con-
trast, the greater swelling of liver slices immersed
in glucose solutions at 37°C and 20°C, as compared
with slices immersed at 4°C, appears to be due at
least in part to the more efficient inward transport,
accumulation and/or metabolism of the immersion
fluid solute (glucose) at the higher temperatures.

321. Gastric secretion in the rat with gastric
fistula and pouch of the entire stomach.
A. C. Ivy, Emma K. Ivy,* E. B. Maacip* anp H.
B. Hayss.* Dept. of Clin. Science, Univ. of Illi-
nois, College of Medicine, Chicago.

In making a chronic gastric fistula in the rat a
small metal flanged cannula was placed in the
rumen of the stomach near the ridge between the
rumen and glandular stomach. When tke rat is not
under study the cannula is plugged. Rats so oper-
ated live indefinitely, one now being alive over 1
yr. postoperative. The pouch of the entire stom-
ach is made as originally described for the dog by
one of us (A. C. J.), a metal flanged cannula being
used to drain the pouch. Such a pouch has been
made with and without section of the vagus
nerves. Such an animal has now survived for over
6 months. Such rats secrete acid gastric juice for

FEDERATION PROCEEDINGS

at least 24 hr. after the withdrawal of food. During§ iif
the 5th hr. after withdrawal of food from the cage fal
the fistula rat secreted 1.2 cc of juice containing § a
3.4 mg of HCl, and the pouch of the entire stomach $f
secreted 1.3 cc containing 2 mg of HCl. During thegt
24-hr. period, the fistula rat secreted 0.3 ce eop.giite
taining 0.8 mg HCl, and the pouch of the entipp§ret
stomach 0.5 ce containing 0.2 mg HCl. Among file
rats with a pouch of the entire stomach with thegil
vagi cut the maximum hourly volume output waite
4.5 cc, and the maximum acidity, 0.501%.

322. Nature of hemorrhagic disorder folloy. we
ing hemolytic transfusion reactions in the
man. Dubey P. Jackson, Jutius R. Krevans*},
AND C. Lockarp ConiEy.* Dept. of Medicine,
Johns Hopkins Univ., Baltimore, Md.

procedure was performed but vaginal bleeding or- gb
curred immediately after the incompatible trans-
fusion. In the other, the first evidence of a trans-Jy,
fusion reaction was the sudden onset of severe
bleeding during a surgical procedure. In both pa-
tients, hypofibrinogenemia, hypoprothrombine-
mia and thrombocytopenia occurred. There was
no evidence of increased fibrinolysis. In addition,
one patient was discovered to have a prolonged] T
clotting time, impaired prothrombin consumption,§y,
reduced Ac-globulin activity, and a low-titered§,
circulating anticoagulant. The fibrinogen and pro-4,;
thrombin returned to normal rapidly but they,
thrombocytopenia persisted for 5-7 days. The co-Syt,
agulation defect that was observed is similar tog,
that produced in experimental animals by thej,
injection of thromboplastic substances. Other,
have demonstrated thromboplastic activity of re
blood cells. It is postulated that the hemorrhagic
disorder that follows hemolytic transfusion reac
tions is due to intravascular coagulation in the
recipient.

323. Adrenocortical function during preg-}.
nancy. JOSEPH W. JAILER, DonALp LONGSON
AND Nicuouas P. Curisty.* College of Physi
cians and Surgeons, Columbia Univ., New Yorke
City.
Several previous investigators have demon-)P

strated that the plasma and urinary corticoid

values increase during pregnancy. The intrave

nous administration of a standardized dose 0

ACTH to 9 women in the 3rd trimester resulted inf

an exaggerated adrenal response, as measured by

the rise in plasma Porter-Silber chromogens.

the nonpregnant state, the plasma corticoids rose
to an average of 45 ug/100 ml as a result of this
procedure whereas in the gravid women the aver

age post-ACTH level averaged 87 ug/100 ml. The K

Yarch 1956

Volume {5

od. During ifference between the two is statistically signifi-
m the cage, § ant- Pretreatment with Prednisone abolished this
containing aaggerated response to ACTH; however, the de-
ire stomach gee Of suppression of the adrenal response was
During thefit a8 great as is found in normals under similar
0.3 ce eon.garcumstances. The levels of plasma corticoids
the entire §rere found to rise in 2 Addisonian patients during
|. Among 3fiie course of pregnancy from pregravid levels of 1
h with thegad 4 ug/100 ml to a height of 19 and 24 ug, respec-
output wyspively. The administration of ACTH was without
%. ect upon these latter values. This indicates that
the hyperresponse to ACTH found in the normal
er follow. pegnant state is presumably of adrenal origin.
ctions int. elevated resting plasma corticoid values found
KREVAns'}, the Addisonian patients are not thought to be
f Medicine, Aine to contributions from the fetal adrenal since
the cord blood contained approximately 60% of
oagulation te maternal level of P-S reactive chromogen. In
ving hemo- dition, the infant’s urinary values for corti-
at received wids, 17-ketosteroids and ketogenic steroids ob-
no surgicalfsined during the first 2 days of life were negli-
leeding oe- ible.

ible trans-
4. Action of guinea pig serum in inhibiting

of a trans-
of severel the growth of a transplantable fibrosar-
n both pa-} coma in rats. ELo1se JAMESON, HERMAN AINIS
hrombine-] yyy R. Mark Ryan (introduced by Pau O.
There was] Greeiey). Univ. of Southern California, Dept.
1 addition,d of Medicine, Los Angeles.
prolonged§ The growth of fibrosarcoma ACMCA2 trans-
sumption,Piented in the AXC 9935 strain of the Irish gray

ow-titerediy; was found to be inhibited by injection of
n and pro-Fuinea pig serum immediately following tumor im-
y but theBintation. Doses of 5 ce, 3 cc or 1 ce were given
8. The eo- mtraperitoneally either daily, on alternate days,
similar tof, every 3rd day, or for 3 consecutive days fol-
Is by thefwed by 3 days rest. The daily injections pro-
-s. Otheriiced comparable results with all 3 doses; how-
vity of redler the dosage was critical when other schedules

morrhagic
sion reac
ion in the’

are used. Five cubic centimeters given on alter-
te days and 5 cc or 3 cc given for 3 consecutive
ys followed by 3 days rest produced the most
hvorable results. When a single injection of 1 cc
a solution of heterologous gamma globulin con-
ining 140 mg/cc was administered intramuscu-
ly in conjunction with daily injections of 5 cc
guinea pig serum, the effectiveness of the guinea
ig serum was decreased slightly. However when
mg of gamma globulin were injected the most
lective inhibition of the tumor was obtained, and
some cases a regression was observed. This is
@ Ist time we have observed regression in this

ng preg-
LoNngGson*)
of Physi
New York

e demon-

corticoid
> intrave
1 dose 0

esulted ing’™°-

asured by

ogens. Ing’ Influence of somatotrophic hormone
coids rose (STH) on organ weights and blood pressure
lt of thisg 2 normal and nephrectomized rats. DarR-
the avery BELL A. Jaques, Ruta B. McVavau, W. F.
0 ml. The Kerrzer anv P. A. RonpeE tu (introduced by

AMERICAN PHYSIOLOGICAL SOCIETY

101

D. F. Bour). Physiology Lab., Univ. of Michigan,

Ann Arbor.

Mature, virgin rats of Wistar strain, weighing
about 200 gm were injected, subcutaneously, with
0.5 mg somatotrophic hormone (STH) per day.
(The hormone was obtained through the cour-
tesy of Armour Labs. and was assayed at 90-100%
the activity of the Armour Standard.) Indirect
blood pressures were taken on alternate days with
a photoelectric tensometer and were compared
with pressures concurrently measured on a group
of untreated animals. Each group comprised 10
animals. STH-treated rats became hypertensive
within 6 days; the mean blood pressure of treated
animals from the 6th to 16th day was 152.0+9.4
mm Hg, the corresponding average for the control
group was 131.7+6.3. Comparison of these values
indicates a statistical confidence level of P = .014.
Mean initial body weights were 204 gm in the con-
trols and 205 gm in the STH-treated group. During
the course of the experiment, control animals
gained an average of 17.5 gm/rat and the treated
animals 33.1 gm/rat. Direct verminal blood pres-
sures recorded from the femoral artery under light
Nembutal anesthesia averaged 118 mm Hg in the
controls and 110 mm Hg in the STH-treated group.
Respiration and sensory-motor response suggested
a lowered threshold for Nembutal anesthesia in
the STH-treated animals. Wet weights of heart,
kidney, gonads and gastrocnemius muscle were
somewhat greater in the treated animals; wet
weights of adrenals showed no difference. In a
parallel preliminary study of the effects of STH in
unilaterally nephrectomized rats the hypertensive
response was variable and inconclusive. Weight
gain was augmented by STH and hypertrophy of
the remaining kidney was marked. (Supported in
part by a grant from the Michigan Heart Assn.)

326. Effects of environmental temperature
and dehydration on susceptibility of mice
to lead poisoning. 8. N. D. JoarpAr* anp A.
M. Baretser. Dept. of Environmental Medicine,
Johns Hopkins School of Hygiene and Public
Health, Baltimore, Md.

The majority of childhood lead poisoning cases
occur in summer. In order to determine if high en-
vironmental temperature and dehydration are re-
sponsible for this seasonal distribution, mice were
exposed to 60°F, 72°F and 95°F environmental
temperatures for 3 days before and 2 wk. after in-
jection with lead. The body temperature did not
average more than +1.5°C from normal. Dehydra-
tion was produced by restricting water intake
leading to 12% loss of body weight over a 3-day
period preceding injection and was maintained
for 3 days after injection. High temperature in-
creased the mortality significantly, hastened the
onset of deaths and accelerated the rate of dying

102

when a solution of lead acetate, sodium thio-
cyanate and sodium citrate was injected intra-
peritoneally and when lead acetate was injected
intravenously. Temperature exerted no effect on
mortality when lead acetate was administered
intraperitoneally, possibly because this substance
formed a precipitate in the peritoneal cavity. De-
hydration significantly increased mortality at all
temperatures and decreased the latent period at
high temperature when either of the lead solutions
was injected intraperitoneally. Exposure to 60°F
had no significant effect on mortality but deaths
continued over a longer period. The effects of high
temperature cannot be explained entirely on the
basis of increased rate of absorption from the peri-
toneal cavity since similar results followed intra-
venous injection; nor do the results parallel body
temperature and expected metabolic changes. The
marked effect of dehydration indicates that rate
of urinary excretion may be an important factor.

327. Vascular responses in the dog’s hind leg
to intra-arterial epinephrine and norepi-
nephrine before and after Dibenzyline.
KENNETH E. Jocuim AND H. G. Ewy.* Dept. of
Physiology, Univ. of Kansas, Lawrence.

In the unopened femoral artery of nembutalized
dogs, pressure was recorded simultaneously with
blood flow recorded with the electromagnetic flow-
meter. Epinephrine and norepinephrine, in doses
of 0.1 and 1.0 ng/kg, were infused into the femoral
artery at a constant rate over a period of 1 min.
before and after administration of 2 mg/kg of Di-
benzyline. Resistance, calculated as the ratio of
mean pressure to mean flow, was plotted as a func-
tion of time. The animals fell into 2 groups ac-
cording to their response to the smaller dose of
epinephrine. In group I, the resistance curve was
M-shaped, with two peaks of constriction; in group
II the resistance curve was W-shaped, with 2
maxima of dilatation. In both groups, the response
to norepinephrine consisted of an initial constric-
tion followed by asmall dilation. This constriction
was generally larger in group I than in group II.
In all animals the effects of the larger dose of each
substance differed only in magnitude and duration
from those of the smaller dose. After Dibenzyline,
group I responded to epinephrine with 1 smaller
constriction followed by a larger dilatation; group
IT exhibited only a single larger dilatation. After
Dibenzyline in both groups the constriction in re-
sponse to norepinephrine was diminished or abol-
ished, and the subsequent dilatation was in-
creased. Evidence indicates that group I and group
IT responses depend on individual differences in
local receptor mechanisms. (Aided by Life Insur-
ance Med. Research Fund and PHS grants.)

328. Excitability changes in anatomical com-
ponents of the monosynaptic arc following

FEDERATION PROCEEDINGS

Volume ij

tetanic stimulation. A. R. Jounson,* P, p,

Watt, J. Y. Lerrvin, W. 8S. McCuuyocu anp¥,

Pitts.* Research Lab. of Electronics, Massathy.

selis Inst. of Technology, Cambridge, Mass.

Using unanesthetized, spinal and decerebrate
cats, nerves from individual muscles were exposed,"
Most experiments were carried out using the
nerves to the gastrocnemius. All ventral roots inf’
the lumbar enlargement were cut. The time courge}
of posttetanic potentiation of a monosynaptic rf
flex was measured. Next stimulating monopolar’.
microelectrodes made of 15 » OD sharpened glass.
covered platinum wire were lowered through sey-f
eral stations into the lumbar enlargement. These!
electrodes were cut off and left in the cord and§
their location was later shown by histological ex.
amination. The change of excitability along the
intramedullary fibers was tested by stimulating
locally with the microelectrodes and recording the
size of the antidromic spike travelling in the rele-
vant sensory nerve. The excitability of tetanized hie
group I fibers from the gastrocnemius is decreased
for a very prolonged time from the root entrances
zone to the region of their endings in the ventral
horn. The excitability of the terminations of non-}
tetanized neighboring group I fibers is not affected tiki
during the prolonged period of depression. Motor
horn cell excitability was tested by direct local}
stimulation and was found not to be changed dur-
ing the long period of posttetanic potentiation. By
(Supported in part by the Signal Corps, the Office
of Scientific Research (Air Research and Develop-
ment Command) and the Office of Naval Research;
and in part by Bell Telephone Labs., Inc.; in parti
by Teagle Fndn., and in part by Natl. Science
Fndn.)

329. Reactions of New Zealand giant rabbits§,,
to ambient temperatures 9°-40°C. Harow
D. Jounson,* C. 8. Coena* anp SAMUEL Bropy.
Dept. of Dairy Husbandry, College of Agriculture,#
Univ. of Missouri, Columbia.

Rabbits resemble European-evolved dairy cat-
tle in being intolerant to temperatures above 27°C
and tolerant to low temperatures; in developing
an explosive rise in respiration rate at 21°C, in
rectal temperature at 27°C, with associated de-
clines in food intake, thyroid activity, milk pro-#
duction; in meeting of skin, hair and air tem-
peratures at 40°C with rectal temperature 41°C.
Rabbit food intake declined from 110 gm at 24°Ogmes
to 3 gm at 37°C. Respiration rates followed am
S-shaped course increasing from 80/min. at 21°C
to 400/min. at 40°C. Pulse rates increased from
100 at 24°C to 150 to 35°C. Skin temperatures inf
creased from 36°C at 21°-27°C environment t
41°C at 40°C. Thyroid activity declined from 1
empirical units at 10°C to 1.2 at 35°C. Rabbits ‘
growing in 28°C chamber had 0.6°C higher rects

L we kA

Volume tj

oN,* PD.
CH AND W,
Massathy.
Vass. *
ecerebrate
"e exposed,
using the
al roots ip
ime course
maptic re.
monopolar
ned glags-
rough sey-
ont. These
cord and
logical ex.
along the
‘imulating
ording the
n the rele-
tetanized
decreased
| entrance
he ventral
ns of non-
ot affected
on. Motor

rect local},

nged dur-
entiation,
the Office
Develop-
Research;
¢.; in part
1. Science

t rabbits
_ Harow
1L Bropy,
griculture,

lairy cat-
rove 27°C
eveloping
21°C, in
iated de-
milk pro-
air tem-
ure 41°C.
n at 24°C

. at 21°C
sed from
atures in

Yarch 1956 AMERICAN PHYSIOLOGICAL SOCIETY 103

ywmperature, 41% lower thyroid activity, 10%
wer heat production, 33% lower food consump-
‘im, 17% lower body weight than those in 9°C
amber. Males were heavier at 9°C, females
yavier at 28°C. At 28°C, mature weight for males
ns 3200 gm, females, 3800 gm; at 9°C, 4400 gm for
inles, 4200 gm for females. Pulse rates at 28°C
jereased from 165 at 50 days old to 135 at 350
ys; at 9°C pulse decreased from 150 to 112 at 50
id 350 days respectively. Respiration rates at
°C increased from 115/min. at 75 days to 125/
nin. at 350 days; at 9°C respiration decreased
om 88/min. at 100 days to 78/min. at 350 days.
Supported by Atomic Energy Commission.)

i). Kinetics of release of radioactive sub-
stances from the frog heart. JoHN A. JOHN-
son. Dept. of Physiology, Univ. of Minnesota,
Minneapolis.
The kinetics of release of radioactive sodium,
ate and sucrose have been investigated in
he isolated perfused frog heart. In general the
netics of release for all 3 substances could be
scribed by a double exponential curve. This was
#t true for all frog hearts, however, and the slope
1some cases seemed to decrease continuously
wking an exponential analysis unrewarding.
the half time for the slow phase of sodium wash-
varied from 19 to 30 min. with the half time
the simultaneous slow sulfate and sucrose
shout always somewhat longer. Whereas the
tal sucrose space agreed in magnitude with that
mputed from the fast sodium space, this was
i true for the total sulfate space. Occasionally
le total sulfate space was larger than the total
mium space. On the assumption that the slow
lease of radioactive sodium represents that
mount coming out of cells the flux of sodium has
#n computed using an average cell fiber diam-
ar of 10 BM.

il. Comparative effects in dogs and monkeys
of sarin and parathion intoxication on
respiratory resistance. RuDOLPH P. JOHNSON
and GusTAVE FREEMAN (introduced by J.
Henry Wits). Chemical Corps Med. Labs.,
Army Chemical Ctr., Md.

Histologic appearances of pulmonary contrac-
le tissue of man and laboratory animals suggest
nsiderable variability in potential pulmonary
nction. Dogs and monkeys under pentobarbital
esthesia were observed following intravenous

Mministration of the cholinesterase inhibiters,

athion and sarin, in doses up to 8 LD5o’s. Tra-
eal airflow and intrapleural pressures were
worded continuously using a pneumotachograph
d strain gauge, and lung-airway resistance was
asured by an interrupter technique during
veral respiratory phases of the intoxication.

iese were stimulatory, pre- and post-apneic

and spontaneous post-apneic gasping. Tidal and
minute volumes were calculated. Early rises in
both tidal and minute volumes, particularly after
parathion, were followed by the usual course for
lethal cholinergic intoxication. Lung airway re-
sistance rose during the post-stimulatory phase
in both species until apnea was attained but was
considerably reduced during the subsequent oc-
casional periods of gasping in monkeys, charac-
terized by high tidal volumes at low respiratory
rates. Resistances attained by dogs were many
times that by monkeys, this being not inconsistent
with the relative quantities of intrapulmonary
muscle. This difference was exaggerated with
parathion as compared to sarin. Greatest resist-
ances were associated with intermediate doses of
inhibitor rather than with 8 LD50’s. Compliance,
work and efficiency were calculated also and will
be discussed. Cardiovascular observations were
made also. Airway-resistances, therefore, follow-
ing lethal doses of parathion and sarin attain
strikingly higher levels in rhesus monkeys than
in dogs and, generally, sarin is more active in this
regard in both species than is parathion.

332. Variations in biliary bile acids, choles-
terol and lecithin in ewes caused by preg-
nancy. CHARLES G. JOHNSTON, HIDEHIKO
SuimurA* AND JoHN F. R. Kuck, Jr.* Dept. of
Surgery, Wayne Univ. College of Medicine,
Detroit, Mich.

Bile acid levels in both hepatic and bladder
bile have been determined in a number of sheep
at the time of cholecystectomy. In ewe hepatic
bile there are 8 mg/ml cholic, 5 deoxycholic and
1.5 chenodeoxycholic acid. Several months later
fistula bile samples were analyzed for the bile
acids, cholesterol and lecithin. A comparison of a
pregnant group with a control group showed the
following differences: in fistula bile of pregnant
sheep the cholic acid concentration was increased
6-fold and the deoxycholic acid concentration was
slightly increased. There were no constant differ-
ences between the 2 groups in the concentrations
of cholesterol, lecithin or chenodeoxycholic
acid. Supported in part by research grants A-659
and A-699 of the Natl. Insts. of Health; the Re-
search Corp. of the Detroit Receiving Hosp., and
the Parke-Davis Co.)

333. Coronary blood flow at 20°C. James R.
JupE, L. M. HaRouTUNIAN AND ROLAND FoLsE
(introduced by Donatp F. Proctor). Dept. of
Surgery, Johns Hopkins Univ., Baltimore, Md.
Coronary blood flow, by the method of Kety

and Schmidt, and other functions noted below

were measured in 11 anesthetized dogs first at
normal body temperature and in 9 of these animals
after cooling to 20°C. Two dogs died of ventricular
fibrillation before measurements were completed.

104

The arterial pH was kept in the normal range
throughout by controlled ventilation. Average
measurements at 20°C expressed as percentage of
the values at normal temperatures are as follows:
coronary blood flow 30%, coronary A-V Oz differ-
ence 82%, myocardial oxygen consymption 26%,
calculated left ventricular work 16%, systemic
A-V oxygen difference 100%, total body oxygen
consumption 24%, cardiac output 21%, peripheral
and pulmonary vascular resistances 300%, coro-
nary vascular resistance 200%. Since the coronary
A-V O, difference is diminished in the cold even
though the calculated myocardial efficiency is
less, we conclude that the coronary blood flow is
sufficient to maintain an adequate supply of oxy-
gen to the myocardium. (Supported by Contract
AF 41(657)-30 with the USAF School of Aviation
Medicine, Randolph Field.)

334. Heat and hypoglycemia in dogs. G. S.
Kanter. Dept. of Physiology, Albany Med.
College, Albany, N. Y.

Recognizing that exposure of dogs to high en-
vironmental temperatures invariably results in an
elevation of their deep body temperature and the
probability that higher temperatures, at least
acutely, speed up metabolism, we became inter-
ested in whether an alteration in body glucose
regulation occurred during such a period. Where
dehydration was allowed to occur, would there be
an increase in concentration of blood glucose as
well as Na and Cl? Exposure of 12 dogs to 120°F
for 4 hr. without access to water resulted in an
average fall in whole blood glucose of 19% and
plasma glucose of 13%, in spite of an average
final dehydration of 5.6% body weight. One would
expect dehydration to cause an increase in con-
centration, yet under the experimental conditions
imposed, hypoglycemia resulted. That the fall in
glucose levels was the result of dehydration per se,
therefore seemed most unlikely. Still, 7 experi-
ments Were conducted, exposing dogs to heat but
maintaining water balance by stomach tube ad-
ministration of water. Here hypoglycemia again
resulted. That the regime of the experiment was
not responsible for the hypoglycemia was tested
in 8 control runs. It appears at present that the
fall in glucose concentration is associated with
the increase in deep body temperature, for when
dogs are exposed to milder air temperatures
(100°F), dehydration but only a slight elevation
in rectal temperature occurs with no fall in glu-
cose levels. A finding of hyperglycemia in man
exposed to desert like conditions has been reported
(in contrast to the hypoglycemia found in dogs),
and may possibly be explained by the presence of
dehydration but absence of elevation of deep
body temperature in the experiments conducted.

FEDERATION PROCEEDINGS

Volume 1§

335. Conductile and integrative functions of
crayfish giant axons. C. Y. Kao* anp §,
Grunprest. Dept. of Physiology and Pharma-
cology, State Univ. of New York, Brooklyn, and
Dept. of Neurology, College of Physicians and
Surgeons, Columbia Univ., New York City.
The diverse nature of some invertebrate central

nervous system components is exemplified by the
ventral nerve cord of the crayfish. In it are four
giant axons, two medial originating in the brain
and devoid of peripheral afferent junctions, and
two lateral originating in peripheral segments and
possessing axono-axonal synapses. The resting
potential of all giant axons is 80-90 mv and the
spike height up to 135 mv. At ca 20°C, spikes last
0.5 msec. followed by a negative after-potential
for about 10 msec. Spikes of lateral giant axons
may be further prolonged by a low amplitude
potential which may persist for 70 msec. This is
superimposed on the repolarization of the spike
and is composed of repetitive and additive synap-
tic potentials. Their existence is correlated with
repetitive spike trains of frequencies up to 400/
sec. following a brief shock, the repetitive re-
sponses often originating at loci other than that
of the initial direct response. Synaptic facilitation
and inhibition are observed. Neither the synaptic
potentials nor the various derived phenomena
occur in the medial giant axons. Thus, the lateral
giant axons differ from the medial in being synap-
tically excitable along their lengths, and resemble
in this respect the segmentally originated giant
axons of the earthworm. In addition to being
conductile, these septate axons, by virtue of the
synapses, possess integrative capabilities diffusely
distributed at segmental levels. The septa them-
selves appear to have no synaptic properties.

336. Respiration during muscular activity in
dogs subjected to hemorrhage. FREDERICK
F. Kao (introduced by ArTrHur A. SIEBENS).
Dept. of Physiology, State Univ. of New York
College of Medicine at New York, Brooklyn.
Pulmonary ventilation, oxygen consumption

and cardiac output (direct Fick) were determined

in 9 normal anesthetized dogs which were sub-
sequently subjected to mild hemorrhage (about

20% of b. wt.). Measurements in intact and bled

dogs were made both at rest and during induced

muscular activity. At rest, ventilation as well as
the ventilatory equivalent for oxygen (VEo:)
increased after hemorrhage. During induced
exercise, the VEo, remained constant in dogs
without hemorrhage whereas in those with hemor-
rhage the VEo, showed a further increase above
the resting (after hemorrhage) level. The oxygen
utilization ratio (O2 consumption after hemor-
rhage/O2 consumption before hemorrhage) de-

cul

olume 1§

tions of
AND H,
Pharma-
lyn, and
ans and
ity.
: central
1 by the
are four
ne brain
ns, and
nts and
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and the
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otential
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iplitude
This is
e spike
) synap-
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rmined
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March 1956

creased precipitously during exercise as a function
of the amount of hemorrhage induced. When the
cdrculatory equivalent for oxygen was plotted
against the oxygen consumption, a curvilinear
relationship was revealed in normal exercised
dogs. The whole curve was shifted parametrically
toward the horizontal axis when these dogs were
bled. The cardiac output increased consistently
during exercise in dogs both with and without
hemorrhage. The ventilation-perfusion ratio in-
creased in intact dogs during exercise, as revealed
by the curvilinear relationship between ventila-
tion and cardiac output; this curve was shifted
up and to the left in dogs with hemorrhage and
exercise. The ventilation-perfusion ratio was also
curvilinearly related to blood pressure in bled
dogs at rest; during exercise this curve was shifted
upward and to the right. (Supported in part by a
research grant (B-458(C2) from the Natl. Inst. of
Neurological Diseases and Blindness, Natl. Insts.
of Health, PHS.)

337. Paper electrophoretic study of the
ground substance and scleroprotein of
connective tissue. Kune-Yine, T. Kao,
Nancy E. Nose anp Rosert J. BovucreK
(introduced by StrepHen W. Gray). Miami
Heart Inst., Univ. of Miami School of Medicine,
Miami, Florida
Protein, carbohydrate and lipid characteristics

of rat connective tissue obtained by sponge-biopsy

technique previously described were investigated

by paper electrophoresis. Tissues of 36 and 120

days of age were successively: 1) homogenized

with saline to separate the ground substance from
the scleroprotein; 2) incubated with hyaluroni-
dase; 3) extracted with 0.1 n NAOH; 4) auto-
claved to solubilize collagen. Protein distribution
per 100 gm of wet tissue is: 4.4 gm, ground sub-
stance; 0.9 gm, hyaluronidase hydrolysate; 0.5 gm,
alkali-soluble fraction and 2.9 gm, soluble col-
lagen. Four protein components were found in the
ground substance with mobilities similar to the
albumin, alpha-1, beta and gamma globulins of
tat serum. A small amount of carbohydrate is
bound at the albumin region and a larger amount
is equally distributed between alpha-1 and beta
globulins. No detectable amount of lipid was
bound with protein-carbohydrate complexes of
the ground substance in contradistinction to the
large amounts of lipids bound to the serum pro-
tein-carbohydrate complexes. The alkali-soluble
fraction consists of a single protein with mobility
similar to alpha-1 globulin. This protein is bound
with both carbohydrate and lipid. Solubilized-
collagen forms a single electrophoretic band in the
beta globulin position. Collagen binds no lipid
and only trace amounts of carbohydrate. A differ-

AMERICAN PHYSIOLOGICAL SOCIETY

105

ence between the electrophoretic patterns of
young and old connective tissue has been observed.
(Supported by Research Grant H-1716, Natl.
Insts. of Health, PHS.)

338. Anticoagulants for turtle blood. HaroLp
M. Kapuan. Dept. of Physiology, Southern
Illinois Univ., Carbondale.

Several common anticoagulants were studied
for turtle blood in vitro to demonstrate that mam-
malian concentrations are unsuitable for tests
such as sedimentation and cell volume in turtle
blood, and to show that there may be set up for
the blood of a given species isotonic solutions
which satisfy the major requirements of a useful
anticoagulant. The packed red cell volumes were
determined for several concentrations of 6 anti-
coagulants. By graphing the packed red cell
volumes against concentrations of each test
chemical and drawing the packed red cell volume
of heparinized blood, used as a standard, as a
horizontal line, the intercept of the test curve
with the heparin line indicated the isotonic con-
centration. Since conditions governing permea-
bility to ions vary continuously in any one cell,
mean intercept values were obtained for each
anticoagulant. Cell size in the test solutions was
checked in 2 dimensions by ocular micrometer
measurements and found by the Student ¢ test
not to vary significantly from that of heparinized
blood. The mean isotonic concentrations (in %)
are: sodium oxalate, 1.376-+-0.089; sodium, 1.455
0.890; potassium oxalate, 1.084+0.198; sodium
fluoride, 1.020+-0.289; lithium oxalate, 0.402+
0.048; and ammonium potassium oxalate, 0.639
0.210. The volume of blood kept fluid by 1 ml of
each anticoagulant in isotonic concentration is,
in the same order as above, 1 to 9; 1 to 9; 1 to 9;
1 to 4; 1 to 8; and 1 to 8.

339. Effects of prolonged exposure of monkeys
to carbon dioxide. 8. A. Kapian, 8. N. STEIN,
R. B. Wiiurams, H. M. Carpenter, J. ANNE-
GERS AND R. E. Lee (introduced by D. J.
Botts). Naval Med. Research Inst., Bethesda, Md.
In order to examine the possible toxic effects of

high concentrations of COs, monkeys were ex-

posed to 3% and 4.5% CO: for periods up to 112

days. The oxygen concentration was maintained

at 21%. In the 3% COs experiment, 10 animals
were exposed to COs, while 10 others, which were
kept at ordinary atmospheric conditions, served

as controls. In the 4.5% COs experiment, 10

animals were kept at ordinary atmospheric condi-

tions for 3 wk. to obtain control measurements
prior to the exposure. The following constituents
of the blood or plasma were measured: CO: con-
tent, pH, pCO2z, NPN, calcium, phosphorus,

106

glucose, sodium, potassium, chloride, bilirubin,
cephalin flocculation, oxygen, hemoglobin, hema-
tocrit, sedimentation rate, white blood cells and
eosinophils. Autopsies were performed on the
animals during or at the end of the exposure pe-
riod. During exposure to 3% COs, the animals
maintained their weights and showed no signifi-
cant alterations in the constituents of their
blood. However, during exposure to 4.5% COs,
they developed anorexia, dyspnea, cough, de-
hydration and loss of weight. The only changes
observed in the constituents of the plasma were
elevation of the CO: content and pCO: and reduc-
tion of the hydrogen ion concentration. Morpho-
logic findings in the group exposed to 4.5% CO,
included 1) a diffuse pneumonitis and 2) hepatic
edema, manifested by intracellular watery vacu-
oles and perisinusoidal edema. The lungs showed
some features in common with the so-called uremic
pneumonitis. The most severe changes were seen
in animals with the most severe respiratory acido-
sis.

340. Effect of warming-up upon physical
performance. PETER V. KARPOVICH AND
CreicHtTon J. Hae.* Dept. of Physiology,
Springfield College, Springfield, Mass.

The few scientific articles related to this topic
are all essentially in favor of warming-up. The
only disagreement concerns the effectiveness of a
preliminary massage. To investigate the effect of
massage, the authors made 60 tests on 7 trained
athletes who were asked to run 440 yd. (outdoors)
after 3 types of warming-up: preliminary exercise,
deep massage and light digital massage. No
statistically significant difference between run-
ning times was observed. To test whether digital
stroking had a psychological warming-up effect,
5 men were asked to run 440 yards (indoors) both
after a digital stroking and also without warm-
ing-up (cold). Each man ran 4 times. There was
no statistically significant difference in running
times, showing that digital massage had no benefi-
cial effect. These 2 series of experiments raised a
question as to the effectiveness of warming-up by
means of exercise. Therefore, an additional test
was conducted. Three men, highly trained, were
asked to ride a bicycle ergometer and do 35 pedal
revolutions at maximum speed (work done was
956 mkg). They did this after a preliminary 5-min.
ride on the ergometer and also without warm-
ing-up (cold). Altogether, 24 tests were made.
No statistically significant difference in riding
times was observed. It seems, therefore, that
warming-up before a sprint-type activity has no
beneficial effect.

341. 17-Ketosteroid determinations in rats:
Method and preliminary results. Seymour

FEDERATION PROCEEDINGS

Volume 15

Katsu (introduced by 8S. R. M. Reynotps),

California Inst. of Technology, Pasadena.

Drekter’s method (1952) has been modified to
allow analyses of small amounts of urine and to
eliminate centrifugation. Conc. HCl, 1.5 ml, ig
added to a 5.0 ml aliquot of a 24-hr. urine sample
in a Pyrex bottle. Place in a boiling water bath for
6 min., then cool. Add 5.0 ml ethylene dichloride
and shake horizontally for 25 min. Add Na laury]
sulphate (5% solution) dropwise to break the
emulsion. Aspirate the top layer and filter the
remainder through Whatman no. 1 paper. Place
1.0 ml of filtrate in 18 X 150 mm test tube and
evaporate to dryness (H2O bath). Standards
using dehydroisoandrosterone in methyl alcohol
are prepared and evaporated. After cooling, 0.4
ml m-dinitrobenzene (1% in methyl cellosolve)
and 0.4 ml of 4.0 n KOH (in methyl! alcohol) are
added. Cover the tubes with aluminum foil and
incubate for 37 min. at 47°C. After cooling, 10
ml of methyl cellosolve are added to each tube
and the resulting color is read at 520 mu. A water
blank and reagent blank are run simultaneously,
Using this procedure the following values ex-
pressed in ywg/24 hr. of 17-ketosteroids excreted
(in terms of dehydroisoandrosterone) by rats
were obtained: female: normal, 180 (+12); hypo-
physectomized, 30 (+9); male: normal, 119 (+17);
hypophysectomized, 41 (+11). Values for the
normal rat agree closely with those in the litera-
ture. Therefore, it seems that the modified Drek-
ter method is useful.

342. Effects of l-norepinephrine and _ 1-epi-
nephrine on coronary flow and oxygen con-
sumption of the intact open chest dog.
L. N. Katz, F. L. Wriutams,* D. Lavurent,*
C. BoLene-Wi.LuiaMs* anp H. Ferrnsere.*
Cardiovascular Dept., Med. Research Inst.,
Michael Reese Hosp., Chicago, Til.

This study was undertaken to elucidate the
effects of the adrenal medullary hormones on
cardiac energetics and coronary vasomotion.
Methods developed in this laboratory were utilized
to prepare 10 dogs for measurement of coronary
flow, myocardial oxygen consumption and mean
arterial blood pressure; cardiac output was main-
tained constant by means of a pump controlling
venous return. After a control period, 5 dogs were
infused continuously with l-norepinephrine (0.5y/
kg/hr.) over a period of 20 min.; /-epinephrine
was similarly given to the other 5 dogs. Observa-
tions were continued after cessation of the drug
to establish a postinfusion control. In all 5 dogs,
norepinephrine infusion consistently effected a
significant rise in coronary flow; in 4 of 5 dogs,
oxygen consumption rose concomitantly. Blood
pressure fell slightly during the administration
of the drug; hence work/oxygen ratio fell in

—

‘ume 15

‘OLDS),

fied to
and to
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ath for
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ak the
er the
. Place
be and
ndards
ilcohol
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il and
ing, 10
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- water
ously,
es ex-
creted
y rats
hypo-
(+17);
or the
litera-
Drek-

1-epi-
1 cOon-
dog.
RENT,*
BERG.*
Tnst.,

te the
es on
otion.
tilized
‘onary
mean
main-
rolling
S were
(0.5y/
phrine
serva-
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dogs,
ted a
dogs,
Blood
ration
ell in

March 1956

every case. Fhe epinephrine response was variable
in our series and no consistent pattern was ob-
grved. Thus the actions of epinephrine and nor-
epinephrine were apparently different under the
conditions of our experiment. The findings with
lnorepinephrine extend the observations pre-
yiously made in this laboratory of an apparent
correlation between oxygen consumption and
coronary flow. They further indicate that l-nor-
epinephrine is capable of eliciting a simultaneous
increase in coronary flow and myocardial oxygen
consumption despite the controlled absence of an
increased work load.

43. Effects of brief interruptions of pure
oxygen breathing upon central nervous
system tolerance to oxygen. B. D. KaurMAn,
§. G. OWEN AND C. J. LAMBERTSEN (introduced
by J. E. Ecxenuorr). Dept. of Pharmacology,
Univ. of Pennsylvania School of Medicine,
Philadelphia.

The convulsions caused by oxygen inhalation
at increased ambient pressures occur only after a
latent period free of signs or symptoms of oxygen
toxicity. In the present investigation the effect
of brief interruptions of oxygen breathing upon
the total duration of oxygen exposure required to
produce toxicity was studied. Twenty-two con-
trol guinea pigs were exposed continuously to
100% O+ at 3.0 atm. At the same pressure, 22 other
guinea pigs breathed 100% O2 for 30 min., 7%
0. in Ne (equivalent to the pO2 of 21% O: at 1.0
atm.) for 10 min., and continued this alternation
for the duration of the experiment. Levels of
inspired CO, did not exceed 0.1% for either group.
In the control guinea pigs exposed continuously
to 100% Oz the first animal developed toxicity at
30 hr., while the mean latent period for the group
was 5.2 hr. The first sign of toxicity in the inter-
mittently exposed group occurred at 9.6 hr.
(oxygen time 7.2 hr.), with a mean time for this
gooup of 17.0 and 12.8 hr., respectively. This
increased tolerance to 3.0 atm. inspired pO2 was
highly significant and indicates that the rate of
recovery from the central nervous manifestations
of oxygen poisoning exceeds the rate of their
development. In addition to extending oxygen
tolerance at increased pressure the alternation of
high and low pO» allowed elimination of nitrogen
during the periods of oxygen breathing and pre-
vented development of ‘bends’ up to 25 hr. total
exposure to 3.0 atm. pressure.

44. Radiosulfate space in tissues. FREDERIC
KAvVALER (introduced by Dayton J. Epwarps).
Dept. of Physiology, Cornell Univ. Med. College,
New York City.

The fact that radiosulfate has a smaller volume
of distribution in man and in the dog than chloride

AMERICAN PHYSIOLOGICAL SOCIETY

107

and chloride-like substances suggested its use in
a reevaluation of the significance of’ the chloride
space in individual tissues. Rabbits were decapi-
tated about 70 min. after an intravenous injection
of radiosulfate. Samples of plasma and of four
tissues (skeletal muscle, liver, heart, and tendon)
were taken for radiosulfate and chloride deter-
minations. Tissue analyses were carried out on
dilute nitric acid extracts of the minced tissues:
chloride by the method of Lowry and Hastings;
radiosulfate as the counting rate obtained from a
barium sulfate precipitate of the extract of con-
stant weight and geometry. Plasma samples were
diluted and analyzed similarly (protein-free fil-
trates were used for the radiosulfate preparations).
Tissue chloride spaces (as percentage of tissue
weight) calculated from these data agreed with
other values obtained in the rabbit. The chloride
space in tendon was found to be about 1} times
the radiosulfate space (70.8%:47.38%). The two
spaces in liver agreed well (18.8:18.7). In skeletal
muscle (13.3:10.2), and in heart (29.3:24.9) the
chloride space was slightly but consistently higher
than the radiosulfate space. If the chloride value
for tendon is taken as representative of that for
all connective tissue, the differences between
chloride and radiosulfate spaces in muscle and
heart might reasonably be attributed to their
content of connective tissue. Calculated on this
basis, the muscle samples were about 5% connec-
tive tissue, the heart samples about 10%.

345. Incorporation of C' formate into free
amino acids and proteins of cell nuclei of
various tissues of the rabbit. Ernest R. M.
Kay (introduced by Witsur K. Smita). Dept.
of Biochemistry, Univ. of Glasgow, Glasgow,
Scotland.

The incorporation of C formate into the
nuclear amino acids and proteins of tissues of the
rabbit has been studied at 2 hr. and 18 hr. after
intramuscular injection of the labeled precursor.
The cell nuclei were isolated by a nonaqueous
procedure (Kay et al. Biochem. J. 62: No. I, 1956).
The cytoplasmic proteins of these preparations
were precipitated with trichloro-acetic acid
(TCA) and the nucleic acids removed, prior to
measuring the activity of the protein. Samples of
the nonaqueous nuclei were extracted with 78%
ethanol and the free amino acids determined by
2 dimensional chromatography. The amino acid
patterns were similar for the different tissues
studied. Radioautographs showed that the ac-
tivity was distributed in all amino acids with
glutamic acid labeled to the greatest extent at 2
hr. The activity of all amino acids was negligible
at 18 hr. Protein fractions were extracted by saline
and separated after dialysis by ammonium sulfate
fractionation. The bulk of the proteins were of a

108

globulin type. Histones and the residual protein
were also prepared. All fractions were carefully
dialyzed where necessary and were precipitated
by TCA and the nucleic acids were removed with
hot TCA. All nuclear proteins had considerable
activity at 2 hr. At 18 hr. the activities were all
reduced. In most of the tissues the residual pro-
tein appeared to be more active than albumin or
globulin. Histone was active at 2 hr. but the
activity was reduced at 18 hr.

346. Variable continued growth in puppies
following total hypophysectomy. A. D.
KeLuerR AND D. M. Wirt.* Physiology Dept.,
Army Med. Research Lab., Fort Knox, Ky.

A variable amount of continued growth has been
encountered in puppies subsequent to total hypo-
physectomies verified by necropsy examination.
No hypophysial rests were found in serial sections
of pharynx. In puppies exhibiting greatest amount
of growth, body conformation was modified with
appearance of ‘Infant Hercules’ tendencies. Long
bones grew in length and rib cartilages ossified
more than in maximal dwarf but less than in
control. Such growth occurred in preparations
exhibiting maximal hypophysial deficits; sexual
infantilism, insulin tolerance reduced 80 times,
atrophied adrenal cortices, marked reduction in
physiological resistance to cold, lowered basal
energy metabolism and complete amelioration
pancreatic diabetes. On other hand maximal
dwarfing has been attained by selective removal
of pars anterior and posterior lobe (ordinary
hypophysectomy). Such a dwarf is actually an
experimental midget because it exhibits no other
hypophysial deficiencies. Sexual development
and function is normal as evidenced by siring
normal puppies. Following pancreatectomy, dia-
betes mellitus is not ameliorated but may be in-
tensified. Responsible variable remains problem-
atical but observations are compatible with
interpretation that maximal dwarfing is due to a
growth-inhibiting factor acting in absence of
pars anterior growth-promoting factor. In absence
of both factors growth proceeds without benefit
of specific endocrine influence. Whether neutrali-
zation of conjectured growth inhibiting factor is
result of removal of stalk tissue (pars tuberalis) or
to involvement of an extra-hypophysial tissue
which is variably impaired by stalk removal pro-
cedure remains an open question.

347. Respiratory response to inspired CO,
during acclimatization to altitude. R. H.
Ke.ioae,* Netto Pace, E. R. ArcHiBaLp*
anp B. E. Vauauan.* Dept. of Physiology,
Univ. of California, Berkeley, and the White
Mountain Research Station, Big Pine, Calif.
The respiratory stimulation produced by CO.

FEDERATION PROCEEDINGS

Volume 1g

was measured in 4 adult males at Berkeley (seq
level) and from 3 hr. to 8 days after arrival at the
Barcroft Laboratory (12,470 ft.). In the test
employed, the subject breathed each of the follow-
ing COz concentrations for successive 10-min,
periods: 0%, 1.1%, 2.38%, 4.7%, 6.4% and (in some
altitude tests) 8.6% COs. Oxygen concentrations
of 21% at sea level and 34% at altitude kept the
inspired Po: at its normal sea level value to elimi-
nate hypoxic stimulation during the tests. The
remainder of each gas was Ne. During exposure
to altitude, the curve relating respiratory minute
volume (BTPS) to the inspired Pecos shifted
markedly to the left, indicating a decrease of
10+3 mm Hg in the Pico, required for any given
degree of respiratory stimulation. The simultane-
ous elevation and steepening of the curve was
relatively slight. Thus the change in CO: response
appears to involve largely a change in ‘threshold’
rather than ‘sensitivity.’ The rate at which the
curve shifted varied widely among the 4 subjects,
but could be reproduced for a given subject on
repetition of the exposure to altitude. The times
required for the shift to become 50% complete
ranged among the subjects from about 6 to 27 hr.
The 90% completion times ranged from about 1 to
6 days. Analysis of the data in terms of the rela-
tion between alveolar ventilation and end-tidal
CO; tension led to similar conclusions. (Supported
by ONR contract.)

348. DNA synthesis and incorporation of
P* in irradiated Ehrlich ascites cells.
Loxa 8. Keuiy (introduced by H. B. Jonzs).
Donner Lab., Univ. of California, Berkeley.

In many mammalian tissues irradiation is
known to depress severely the incorporation of
precursors into DNA (e.g. KE.ty et al. Rad. Res,
2: 490, 1955). However, several investigators have
found that carcinomas show relatively little
depression and have suggested that the incorpora-
tion is due to turnover of existing DNA not ac-
companied by net synthesis (e.g. ForssBERG
AND K.ie1n. Exptl. Cell Res. 7: 480, 1954). The
experiments to be reported were performed to test
this possibility. All measurements were made 46
days after the inoculation of approximately 2X10!
Ehrlich ascites tumor cells. The 2-hour incorpora-
tion of P-32 into DNA was measured at various
times after irradiation. With a radiation dose of
800 r no significant depression in DNA specific
activity was observed until 24 hr. Measurements
of average cell volume, total nucleic acid per cell,
and DNA per cell at 12 and 20 hr. revealed that
all three increased at the same rate, closely
matching the growth rate of the unirradiated
tumor and the rate of DNA formation estimated
from incorporation data. At 48 hr. after radiation,
cell volume and total nucleic acid per cell had

pol:
ett,

inte

lume 15

ey (sea
] at the
he test
follow-
10-min.
in some
Tations
ept the
o elimi-
ts. The
Xposure
minute
shifted
ease of
y given
ultane-
ve was
esponse
‘eshold’
ich the
ibjects,
ject on
e times
mplete
> 27 hr.
rut 1 to
1e rela-
\d-tidal
yported

on of
cells,
JONES).
ley.
ion is
tion of
ud. Res,
rs have
- little
orpora-
not ac-
SSBERG
'). The
to test
ade 4-6
2X10"
orpora-
various
dose of
specific
ements
er cell,
ad that
closely
adiated
imated
liation,
ell had

March 1956

risen even litgher while DNA per cell showed little
further increase. The maximum DNA content
reached was approximately the octoploid value.
The data suggest that the irradiated cells con-
tinue to synthesize DNA until they reach the
premitotic DNA content (octoploid in the Ehrlich
cells) and are arrested there because they are
uable to go through mitosis.

349. Application of the polarographic method
for continuous recording of oxygen tension
with platinum electrodes. ALBERT F. KELso
AND CLARENCE N. Petss (introduced by ARTHUR
H. Srernnaus). Dept. of Physiology, Stritch
School of Medicine, Loyola Univ., Chicago, Ill.
A method for continuous recording of tissue

oxygen tension is being investigated. A modified

polarographic method developed by Olson, Brack-

ett, and Crickard (J. Gen. Physiol. 32: 681, 1949)

permits recording of oxygen tensions at 10-sec.

intervals in solutions with continuously changing
oxygen tensions. A plastic diffusion cell has been
constructed for measurement of oxygen diffusion
coefficients for various membranes and solutions.

The diffusion cell is suspended in a constant tem-

perature bath and the temperature of the cell

solutions is monitered against the bath tempera-
ture by means of a thermocouple. The cell con-
sists of two chambers separated by a membrane;

a potential chamber containing a solution with

constant oxygen tension, and a diffusion chamber

in which oxygen tensions are measured by static
platinum electrodes located at multiple points
along the axis. The chambers are separated by
prepared or selected membranes, and the oxygen
tension of the potential chamber is monitored at
the membrane surface. Electrodes are calibrated
against solutions with known oxygen tensions.

Variation for a given electrode during continuous

recording at a constant oxygen tension is less than

1%. Over a period of 1 month variation for a

given electrode is less than 5, and generally less

than 1%. Diffusion coefficients of the membrane
and diffusion chamber solution are calculated
from the rate of change in oxygen tension meas-
wed at the electrodes in the diffusion chamber.

Discussion of results will include the application

to tissue recording.

0. Effects of epinephrine and norepineph-
rine before and after Dibenzyline in dogs
with a mechanical heart. J. E. Kenpricx
(introduced by G. N. Loorsourrow). Dept. of
Physiology, Univ. of Kansas, Lawrence.

The effects of epinephrine and norepinephrine
before and after Dibenzyline were studied in dogs
in which the heart was replaced by a double
piston-type pump which allowed the ‘cardiac
output’ to be kept constant at any desired value.

AMERICAN PHYSIOLOGICAL SOCIETY

109

Before Dibenzyline, 10 ug/kg of epinephrine pro-
duced, in 9 animals, an average pressor response
of 36% above the control blood pressure level.
Ten ywg/kg norepinephrine caused an average rise
of 35%. Two ug/kg doses of epinephrine and nor-
epinephrine caused an average rise in pressure of
12 and 13%, respectively, in these animals. After
injections of Dibenzyline ranging from 0.5 to 5.0
mg/kg (14 animals), the pressor response to nor-
epinephrine in all doses used was diminished or
abolished but never reversed. The pressor response
to 10 wg/kg epinephrine was either abolished or
reversed after a total dose of 3 mg/kg or more of
Dibenzyline. The pressor response to 2 yg/kg
epinephrine was more often abolished than re-
versed, even after the larger doses of Dibenzyline.
These results indicate that, in a dog with a con-
stant cardiac output provided by a pump, both
epinephrine and norepinephrine increase the total
peripheral resistance and to approximately the
same extent. Dibenzyline can abolish the con-
strictor effect of both. Norepinephrine, under the
conditions of these experiments, exhibits little
or no vasodilator action as contrasted with
epinephrine. (Author is Public Health Research
Fellow of the Natl. Heart Inst.)

351. Diuretic properties of compounds of
mercury. R. H. Kessiter* anp R. F. Pitts.
Dept. of Physiology, Cornell Univ. Med. College,
New York City.

Utilizing the radioactive isotope, Hg?*, to
facilitate measurement of distribution and urinary
excretion, a group of organic compounds of mer-
cury are being studied with a view to defining
those structural characteristics which determine
diuretic activity. To date we have investigated
3-chlormercuri-2-methoxy-propyl urea (Chlor-
merodrin), mercuric chloride, parachloromercuri-
benzoate and methylmercurichloride in doses of
1 and 2 mg. Hg/kg b. wt. in anesthetized dogs
previously hydrated by the administration of a
liter of saline. One kidney has been exposed in
the flank and a Geiger tube placed in contact
with its surface to measure qualitatively the
accumulation of mercury in the outer cortex. In
addition, G.F.R., sodium, water and mercury ex-
cretion, and at the end of the experiment, tissue
distribution have been measured. Only Chlor-
merodrin and mercuric chloride have exhibited
diuretic properties. These compounds are highly
and rapidly concentrated in the renal cortex and
rapidly excreted in the urine, the former 50 and
the latter 20% excreted in 2} hr. Parachloro-
mercuribenzoate is rapidly excreted (35% in
24 hr.), but much less concentrated in the renal
cortex. Methylmercurichloride is but little con-
centrated in the kidney and very slowly excreted.
Although the latter 2 compounds are excellent

110

inhibitors of sulfhydryl enzymes in vitro, they are
not diuretics as we have used them.

352. Left atrial and systemic arterial dilution
curves after injection of indicator into left
and right heart of humans. Joun R. Keys,*
H. J. C. Swan, Peter S. Herze.* anp Ear
H. Woop. Section of Physiology, Mayo Fndn.
and Mayo Clinic, Rochester, Minn.

During simultaneous catheterization of the
left and right sides of the heart, indicator-dilution
curves were recorded after injection of T-1824
into the superior vena cava (SVC), pulmonary
artery (PA) and left atrium (LA). Samples of
blood were drawn simultaneously through cuvette
oximeters located at the radial artery (ra) and
the left atrium (la). Injection sites are designated
by capital letters and sampling sites by small let-
ters as subscripts. Five patients with mitral steno-
sis or aortic stenosis, or both, were studied. Their
hemodynamic data varied considerably. Cardiac
indices ranged from 1.4 to 4.2 (av. = 2.3) 1/min/
m?*, With the exception of PT), curves, the car-
diac output calculated from curves at various
sites compared well with each other and with the
Fick determination. The average appearance
times in seconds were: LAra, 5.4; PAia, 2.7; PAra,
10.3; SVCia, 6.38; SVCra, 14.1. The seconds required
for the average particle to reach the sampling
site (mean transit times) averaged as follows:
LA, 123; PAy, 79; PAs, 21.4: SVC,,;. 188;
SVC,., 29. Recirculation times were nearly iden-
tical at the various sites in an individual patient
(av. = 34.1 sec. from all sites). The average cal-
culated blood volumes (in ml) were: LA;sa, 680;
PTj,, 450; PT +a, 1190; SVCis, 720; SVC ra, 1670.

353. Responses from auditory cortex to re-
peated bursts of noise. NELSON YUAN-
SHENG Krianc AND Morse H. GoupsteEIn, JR.
(introduced by S. S. Stevens). Massachusetts
Inst. of Technology, Cambridge, Mass.
Responses to repeated clicks and bursts of noise

were recorded with gross electrode from the audi-
tory cortex and from a location near the round
window of the cochlea in anesthetized cats. The
repetition rate was varied from less than 1/sec.
to beyond 1000/sec. As the repetition rate in-
creases the evoked cortical responses decrease in
size and become visually undetectable at approxi-
mately 20/sec. Summated neural potentials from
the peripheral location ‘follow’ to rates of at least
several hundred clicks or bursts/sec.

354. Ethanol metabolism in the rat. F. W.
Kinarp, J. C. Auti* anp W. J. Roperts, Jr.*
Depts. of Physiology and Chemistry, Med. College
of South Carolina, Charleston.

The purpose of this study was to make a com-
parison in the same animal of two different tech-

FEDERATION PROCEEDINGS

Volume if

niques in determining the rate of ethanol me-
tabolism, e.g., determination of the declining
concentration of blood alcohol with time and the
direct determination of ethanol in the ground,
mixed tissues at a given interval following ad-
ministration of a known quantity of alcohol,
In general the rate of alcohol metabolism was
lower and the factor r higher when calculated from
the declining blood values than when determined
directly. The directly determined r was greater
than the sum of water and ether-extract of the
tissue. It appears that errors inherent in the Q
value may explain some of these discrepancies,
(Supported in part by a grant from the Com.
mittee on Problems of Alcohol, Nat. Academy of
Sciences-Natl. Research Council.)

355. Effect of uncompensated respiratory
acidosis on carbohydrate metabolism as
related to adrenal function. C. T. G. King
(introduced by R. Greer). U. S. Naval Med,
Research Lab., U. S. Naval Submarine Base,
New London, Conn.

Studies on liver, muscle glycogen and blood
sugar were carried out in normal, hypophysec-
tomized and adrenalectomized mature male rats
of the Wistar Hisaw strain, which were rendered
acidotic by exposing them to an atmosphere of
30% carbon dioxide in oxygen for periods of up to
2 hr. and recovery of 1 hr. in air. The liver glyco-
gen content of the normal and hypophysectomized
animals was significantly decreased during the
first 10 and 30 min. of exposure, but at 1 hr. the
values returned to normal and remained so if the
rats were followed to recover on air. When the
exposure continued for 2 hr., the liver glycogen
values fell significantly and again returned to
normal on air. The liver glycogen of the adrenalec-
tomized animals fell consistently during exposure
and recovery. Without indications of a return to
normal, muscle glycogen did not change drasti-
cally during exposure in any of the experimental
groups but in all cases was below normal at re-
covery. An acute hyperglycemia developed in
normal and hypophysectomized rats during the
exposure with a return to normal during recovery.
The adrenalectomized animals gave evidence of
hyperglycemia (20% increase) at 10 min. of expo-
sure but after 1 hr. went into a hypoglycemic state
from which they did not recover even after transi-
tion to air.

356. Biosynthesis of adrenaline and _ nora-
drenaline by adrenal slices. NoRMAN KiRsx-
NER AND McC. GoopaAtt (introduced by KEITH
S. Grimson). Dept. of Physiology, Duke Univ.
Med. School, Durham, N. C.

Adrenal slices incubated with C™ labeled tyro-
sine form C™ labeled adrenaline (A), noradren-
aline (N) and hydroxytyramine (H). Incubations

olume 15

nol me-
leclining
and the
ground,
ying ad-
alcohol.
ism was
ted from
ermined
greater
t of the
1 the C
pancies,
ie Com-
demy of

iratory
ism as
x. Kine
al Med,
e Base,

d blood
yphysec-
ale rats
endered
dhere of
of up to
r glyco-
tomized
‘ing the
hr. the
30 if the
hen the
rlycogen
rned to
renalec-
xposure
turn to
. drasti-
‘imental
il at re-
oped in
ring the
covery.
lence of
of expo-
nic state
r transi-

| nora-
| Krrsu-
y Keita
te Univ.

ed tyro-
oradren-
ubations

March 1956

were carried out in Krebs’ phosphate buffer
(Biochem. et Biophys. Acta 4: 249, 1950) containing
either glucose or glucose-6-phosphate. Adrenaline,
noradrenaline and hydroxytyramine are initially
separated from tyrosine by absorption on a 2.5 X
0.9 cm column of amberlite IRC-50. After elution
with 2.0 m acetic acid (HAC) the amines were
separated from each other on a 35 X 0.9 em column
of IRC-50 buffered at px 6.1 with 0.2 m NH,AC;
dution with 0.4 m NH,AC at pu 5.0 effects the
separation. Fractions (1.5 ml) were collected at a
fow rate of 3 ml/hr. Recovery of adrenaline,
noradrenaline and hydroxytyramine varies from
"%-95%. In a typical experiment 0.2-0.3 gm
of adrenal slices were incubated with 1 xX 10°
counts/min. of uniformly labeled C** tyrosine
(§.A. 7.7 me/mm) for 3 hr. at 37°C. A gas mixture
containing 93.5% Os and 6.5% COs was bubbled
through the medium; the volume was 3 ml. CPM
in the isolated fractions were: A, 1242; N, 14,910;
H, 3442. The identity of the fractions was checked
by paper chromatography and recrystallization to
constant specific activity. (Aided by Natl. Inst.
of Health grant H-2140(C).)

37. Nature of C'* compounds recovered in
portal plasma during absorption of C1*-
labeled hexoses. J. Y. Kryasu* anv I. L.
Cuarkorr. Dept. of Physiology, Univ. of Cali-
fornia School of Medicine, Berkeley.

A method for the complete collection of portal
blood draining an isolated in situ jejunal loop is
described. By the use of this technique and chro-
matographic analysis, the quantitative distribu-
tio of C4 compounds in portal plasma has been
determined during absorption of C' hexoses by
tats and guinea pigs. Experiments with evenly
labeled glucose in the rat showed that about 90%
of the C4 in portal plasma was present as such
and the remainder as lactate and alanine (Bio-
thim. et biophys. acta. In press). Experiments
with evenly labeled fructose in the rat showed that
about 50% of the Cin portal plasma was present
48 fructose and approximately 40% as lactate and
other metabolites. About 10% of the C4 was found
as glucose. In the guinea pig fed fructose-C%,
about 30% of the C in portal plasma was present
48 fructose, 10% as lactate, and 60% as glucose.
Thus a sizable portion of absorbed fructose enters
the liver as fructose.

88. Isolation of purified human plasmin
(fibrinolysin). Danret L. Kiine anp JAcoB
B. Fisuman.* Dept. of Physiology, Yale Med.
School, New Haven, Conn.

Starting with purified human plasminogen,
prepared by a method described previously (J.
Biol. Chem. 204: 949, 1953), plasmin has been pro-
duced by streptokinase (SK) activation, and a
stable, active fibrinolytic protein has been iso-

AMERICAN PHYSIOLOGICAL SOCIETY

111

lated by alcoholic fractionation. Approximately
5500 u of SK (Varidase, Lederle) per mg of plas-
minogen was added to a solution containing 4
mg/ml of the enzyme at px 6.8. After incubation
for 10 min. at 37.5°C, the solution was adjusted
to px 8.7 and centrifuged. The px was then lowered
to 7.6 and again centrifuged. At 0°C, cold 95%
ethyl alcohol was added to a final alcohol concen-
tration of 21% by volume. The precipitated plas-
min was collected by centrifugation after 16 hr.
at 4°C and was dissolved in distilled water. In
the frozen state, this preparation still retains its
original activity after 7 months. The plasmin so
obtained was 2-5 times more active per mg of ni-
trogen than the unfractionated solution when
tested against a standard fibrin clot. The bovine
fibrinogen used in the clot was free of bovine
plasminogen. The addition of 1000 u of SK/mg
N to each assay tube did not affect the activity
of any of the fractions. Preliminary in vivo studies
in the dog showed rapid dissolving of a clot after
the intravenous injection of 0.5 mg plasmin/kg.
Preliminary toxicity studies being conducted in
association with Dr. L. Nahum revealed no sig-
nificant changes in blood pressure, electrocardio-
gram or body temperature after the intravenous
injection of up to 1 mg of plasmin/kg in the dog.
Two ml! of human blood failed to clot when added
to 0.1 mg of plasmin.

359. Physiological effects of growth hormone
of primate origin in the hypophysectomized
rhesus monkey. Ernest Knosit anp Roy
O. Greer. Dept. of Physiology, Harvard Med.
School and Biological Research Lab., Harvard
School of Dental Medicine, Boston, Mass.
Previous studies have shown that beef and pig

growth hormone preparations when administered

to normal, hypophysectomized and _ partially
pancreatectomized monkeys had no significant
effect on growth and various aspects of protein
and carbohydrate metabolism. Similar equivocal
findings have been reported for man. The avail-
ability of several thousand monkey pituitary
glands made possible the preparation of alkaline
extracts which, as determined by rat assay, were
rich in growth hormone activity. The adminis-
tration of these extracts to hypophysectomized
rhesus monkeys produced a striking elevation in
fasting blood sugar, a decrease in glucose toler-
ance, and a decrease in insulin sensitivity. Iden-
tically prepared alkaline extracts of beef pitui-
tary glands were ineffective in this regard. A
sample of purified monkey pituitary growth hor-
mone prepared by Raben and Astwood produced
alterations in carbohydrate metabolism similar
to those observed when the alkaline monkey pitui-
tary extracts were administered. Purified beef
growth hormone prepared by the same procedure

112

was ineffective. Studies dealing with the effects
of monkey pituitary growth hormone on nitrogen
balance in normal and hypophysectomized mon-
keys are currently in progress. It appears from
the above that in contrast to most other anterior
pituitary hormones, growth hormone exhibits
characteristics of species specificity and that the
apparent ineffectiveness of beef and pig growth
hormone preparations in primates can probably
be explained on that basis.

360. Uterine myosin. D. R. Kominz anp F.
Saap (introduced by C. I. Wrieut). Natl. Inst.
of Arthritis and Metabolic Diseases, Natl. Insts.
of Health, Bethesda, Md.

To clarify the question whether myosin or a
labile complex of actin and tropomyosin is the
contractile protein in myometrium, ultracentrif-
ugal sedimentation studies have been performed
on various extracts of human uterus. Rich yields
of myosin B are obtained if ATP is employed in
the extracting medium; if ATP is omitted, there
is much less myosin B extracted so that the ultra-
centrifuge pattern shows impurities such as tropo-
myosin becoming quantitatively more significant.
Amino acid analyses also have been performed on
purified tropomyosins from skeletal, cardiac and
uterine muscle. Whereas the tropomyosins from
the striated muscles are identical, uterine tropo-
myosin differs from them in aspartic acid, histi-
dine, lysine, arginine, proline, methionine, iso-
leucine and tyrosine. Reflecting these differences,
uterine tropomyosin crystallizes as needles in-
stead of hexagonal plates. These studies indicate
that the difference between the myosins of striated
and smooth muscle is not to be found in the molec-
ular complex postulated by Snellman and Tenow
(Biochim. et biophys. acta 13: 199, 1954) but is prob-
ably to be found in submolecular structural
differences such as are found in the amino acid
composition of the tropomyosins.

361. Simultaneous determination of red cell
volume using radioiron and radiochromate.
Leon Krarntz,* E. L. Smita anp R. A. Hue-
eins. Univ. of Texas, Dental Branch and Baylor
Univ. College of Medicine, Houston.

By taking advantage of the differences in the
gamma-ray energies of radioiron (Fe®) and radio-
chromium (Cr®!) it is possible to differentiate be-
tween the 2 isotopes in the same sample. Using a
well-type scintillation counter, the same sample
is counted at 2 different voltage settings of the
photomultiplier tube. By counting a separate
radiochromium and radioiron standard one de-
termines the voltage at which only radioiron and
no radiochromium is detected. Raising the voltage
to the usual operating voltage of the instrument,
both of the isotopes are detected. By multiplying
the ratio of iron counts at the 2 voltage settings

FEDERATION PROCEEDINGS

Volume 15

times the iron counts at the lower voltage, the
amount of radioiron in a mixed sample at the
higher voltage can be calculated. Dogs given
radioiron several weeks prior to the experiments
were used as the source of radioiron-labeled cells,
Acute blood volume and red ecli volume studies
were done on some of these dogs by additional
labeling of an aliquot of their own blood in vitro
with radiochromate. Control studies were done
in other dogs using double-labeled cells. Alternate
determinations were made in the same dogs
using one isotope initially and the other ter-
minally. Similar studies were done in dogs sub-
jected to massive transfusions. In general, the
results obtained for red cell volumes and blood
volumes were in good agreement when the isotopes
were used separately or simultaneously.

362. Effects of glycyrrhiza on salt and water
retention in the rat. Sarrtey D. Kraus anp
Lewis H. Kurrz (introduced by Watter M.
Booker). Dept. of Pharmacology, Howard Univ,
Med. School, Washington, D. C.

An interest in the place of glycyrrhiza and its
derivatives in clinical medicine arose in Holland
in 1950 when a DCA-like action of sodium and
water retention and potassium excretion was
observed in both normal subjects and patients
with Addison’s disease. Despite reported success
of glycyrrhiza as a substitute for DCA in adrenal
insufficiency, substantiating evidence in the in-
tact and adrenalectomized animal has been lack-
ing. The study of the effect of subcutaneous ad-
ministration of glycyrrhiza on urine and sodium
output of intact male rats during 4-hr. collection
periods was undertaken as a basis for future in-
vestigative work on the mode of action of gly-
cyrrhiza. The daily administration of 20, 40 and
80 mg doses for 5 consecutive days produced
sodium retention after a 2-day latent period which
had a linear response to dose. An antidiuretic
effect was apparent immediately. Withdrawal of
treatment resulted in a rebound excretion of
retained extracellular sodium before a normal
state was re-established.

363. Tyrosine oxidizing system of the liver of
young and fetal rats. NorMAN KRETCHMER
AND HELEN McNamara (introduced by HENRY
L. Barnett). Dept. of Pediatrics, The New York
Hosp.-Corneil Med. Ctr., New York City.
With manometric procedures it was determined

that fetal rat liver had a tyrosine oxidizing ac-

tivity of 3 ul O2 uptake/30’/mg N. This was com-
pared to the activity of the newborn rat liver of

35 ul O2/30’/mg N. and 50 ul 02/30’/mg N. in the

adult liver. Five hr. after birth the activity of the

tyrosine oxidizing system fell to 20 ul O2/30’/mg

N. The liver of litter mates of the same age who

were not nursed had an activity of 35 ul 02/30’/mg

lume 15

ge, the
at the
S given
riments
ad cells,
studies
ditional
in vitro
re done
lternate
1e dogs
ner ter-
gS sub-
ral, the
d blood
isotopes

1 water
\US AND
‘TER M,
rd Univ.

and its
Holland
um and
ion was
patients
success
adrenal
the in-
en lack-
20us ad-
sodium
ollection
ture in-
of gly-
, 40 and
roduced
»d which
idiuretic
rawal of
etion of
normal

liver of
=’ TCHMER
y HENRY
Tew York
City.

-ermined
izing ac-
yas com-
, liver of
Y. in the
ty of the
o/' 30'/ mg
age who
2/30’ /mg

March 1956

N. When tlie 60-min. oxygen uptake curves ob-
tained with these livers were compared, it was ob-
grved that the curves derived from the livers of
the non-nursed animals resembled those of the
livers of newborn rats of the same litter. The
curve obtained with the livers of the nursed ani-
mals is depressed after 10 min. and maintains a
much lower value during the rest of the 60-min.
priod. It was also found that the tyrosine trans-
aminase apoenzyme is 10 times more concentrated
in the adult rat liver than in the fetal rat liver.
Transamination is the first and obligatory step
in tyrosine oxidation, thus the lack of apoenzyme
isat least in part responsible for the minimal tyro-
sine oxidizing activity of fetal liver.

%4. Determination of oxygen diffusion coeffi-
cients in hemoglobin solutions of different
concentrations. F. Kreuzer (introduced by
Roy P. Forster). Dept. of Physiology, Univ.
of Fribourg, Switzerland.

Numerous photoelectric measurements have
been made on the rate of penetration of oxygen
into very thin layers (50-500 u) of hemoglobin solu-
tions by Pircher and Kreuzer, covering the whole
concentration range of Hb from 0.03 to 40 gm%.
Previously reliable calculations of Do, were only
possible from observations made on 0.03 gm%
Hb solution, for only in such dilute solutions the
themical reaction of oxygen with Hb does not
tause appreciable complication since the fraction
df oxygen combining with Hb is small compared
with that in physical solution. Somewhat less
tliable calculations could be made with Hb in
the range of 1-8 gm%. At high concentrations,
ie. 35 gm% Hb and above, the ‘advancing front’
formula previously used by Hill and by Longmuir
ad Roughton (for CO and Nz) can be applied.
Klug and Roughton calculated values of Do,
fr this high concentration range, which is of
prime physiological importance, from the author’s
lata (final paper by these 3 authors, now in press).
It was found that Do, = 3.5-10- cm?/sec. for
%.5 gm% Hb at 25°C. These results agree quite
vell with the Dco and Dy, as determined by
longmuir and Roughton. In the middle range Hb
ind oxygen diffusions are both limiting, and no
tally satisfactory theoretical method is available.

45. Vitamin A, carotenoids and plasma lipo-

proteins. Norman I. Krinsky, Davin G.
CoRNWELL AND J. L. OnciEy (introduced by
D. R. Grirrin). Biological Labs., Harvard Univ.,
Cambridge and Dept. of Biological Chemistry,
Harvard Med. School, Boston, Mass.

We have undertaken an investigation of the
techanism of transport of vitamin A alcohol, and
iquantitative study of the distribution of carot-
noids in the various lipoprotein fractions of
luman plasma. Pooled plasma was subjected to

AMERICAN PHYSIOLOGICAL SOCIETY

113

several fractionation procedures, including pre-
cipitation with dextran sulfate and ultracentrif-
ugal flotation, to obtain distinct lipoprotein
classes. Chromatographic analysis of the carot-
enoids obtained from plasma and its fractions
indicated that 3 main carotenoids are present;
B-carotene (plus traces of a-carotene), lycopene
and lutein. Approximately 90% of the total
plasma carotenoids are distributed between the
a- and £-lipoprotein fractions. Only traces of
vitamin A are found in the f-lipoprotein precipi-
tated with dextran sulfate. The a-lipoproteins
remaining after this treatment contain 10-15% of
the total plasma vitamin A. When plasma is sub-
jected to high-speed centrifugation in a high-
density medium, during which the low-density
proteins float, 80-90% of the vitamin A is found
with the non-floating proteins. It is concluded that
vitamin A is transported in human plasma asso-
ciated with a protein or proteins other than the
a- and £-lipoproteins.

366. Intracellular recording of cortical poten-
tials. K. Krnsevié (introduced by E. A. Boyr-
DEN). Dept. of Physiology and Biophysics, Univ.
of Washington School of Medicine, Seattle.

In order to record more than transient intra-
cellular potentials from injured cells, it is essen-
tial to abolish cerebral pulsations. This can be
done by perfusing the head of the cat at a steady
pressure. Blood from the descending aorta is
pumped by the heart into a bottle against an ade-
quate air pressure. From there it flows at an even
pressure into the head via a warming coil and
the innominate artery. A level sensitive device
inside the bottle balances the flow from the aorta
with the flow into the head by small and slow
compensatory variations of air pressure. There
are provisions for monitoring throughout the
experiment perfusion pressure, flow and blood oxy-
gen tension, and also for the injection of test solu-
tions into the perfusing system. It has been pos-
sible by this method to maintain the cortex in a
viable condition for periods of up to 6 hr. Glass
ultramicroelectrodes were inserted into the cortex
after removal of the pia; this allows free entrance
of even the finest electrodes without breakage or
dimpling. (Supported by Grant B-396(C2) from
the Public Health Service.)

367. Use of a relaxin reference standard for
bioassay of extracts in estrogenized intact
guinea pigs. Rosert L. Kroc, Vivian L.
Braco AND NEIL R. Srastuui (introduced by
Artuur A. Heuipaum). Warner-Chilcott Re-
search Labs., Morris Plains, N. J.

An extract in stable powder form prepared from
hog ovaries has been designated for use as a re-
laxin reference standard. Virgin female guinea
pigs, 325-400 gm at start, are primed weekly with

114

estrogen and the Standard. The degree of pelvic
mobility is estimated and scored by manual pal-
pation. Only pigs whose scores are classified as
‘positive’ are eligible for assay use each week
thereafter. ‘Unknown’ preparations are adminis-
tered to groups of 10 or more pigs in parallel with
the Standard at two or more dosage levels. Pigs
showing a ‘negative’ response are primed with the
Standard (and estrogen) the following week(s).
Bioassay data on relaxin preparations including
vialed lots of Releasin will be presented. Two of
the lots are control preparations—one made from
the reference Standard powder and one from a
low-potent stock powder. The data indicate that
acceptable relative potency estimates may be
obtained with a minimum of 80 animals equally
distributed between Standard and the ‘unknown’
preparation.

368. Sine wave expressions of respiratory
phenomena. Huco Krurcer. Depts. of Ani-
mal Husbandry and Zoology, Oregon State College,
Corvallis.

Simplicity of expression may sometimes be
gained by the formulation of respiratory (and
other periodic biological) phenomena as sine
curves. During the approximate steady state
known as basal, the periodic aspects of the volume
V of dead space plus alveolar air may be expressed
by V =f (@) =f (+ T), where ¢ is time and 7
is the period in minutes. If F is the respiratory
frequency per minute, 7-F = 1. The sine wave
character of the respiratory phenomena may be
formulated by V = Vo + A-Sin 2x Ft, where Vo
is the ml of air in the alveoli and dead space at
the midinspiratory point, 2A is the tidal air in
ml, and F is the respiratory frequency. Body
weight W can be expressed as W = Wy — Bi +-Ad-
Sin 27 Ft, where Wo is the weight at a given mid-
inspiratory point, B the metabolic and water
weight loss with time, and d is the density of the
inspired air. If the elastic recoil of the lungs has a
value of 10 cm of water in the basal expiratory
position, and the tidal volume is 500 ml, the elas-
tic recoil E of the lungs during the respiratory
cycle is given by E = 10 + 4.5Q, where Q is the
increase in lung volume in liters.Q = 0 when V =
Vo + 250 and ¢ is 1/4F; with a respiratory fre-
quency of 12, this will be when ¢ = 1/48 min. (1.25
seconds). Generally, Q = 0.250 — 0.001A sin 2zF t;
E = 10 + 4.5(0.250 — 0.001 X 250 X Sin 27F 2%);
E = 11.25 — 1.125 sin 2xF t. E will vary between
10.0 and 12.5 cm of water. (See Ronrer. Bethe’s
Handbuch, 1925.)

369. Blood chemical differences between
Hereford and Angus cattle. Huco KRvEGER,
M. A. MacDonatp* aNnp RatpH Boagarrt.*
Dept. of Animal Husbandry, Oregon State Col-
lege, Corvallis.

FEDERATION PROCEEDINGS

Volume 15

Of the 4 categories (Hereford and Angus males
and females) Hereford males were unusual jp
having the lowest hemoglobin average at 5
and 800 lb. b. wt.; a low uric acid and the lowest
amino acid average at 800 lb., and the highest
urea at 800 lb. Hereford females were unusual in
having a high uric acid average at 500 lb. anda
low uric acid average at 800 lb.; they possessed
the lowest creatinine average at 500 lb. and the
highest at 800 lb.; they had the highest glucoge
average at 800 lb. and were the only group to
show an increase in blood creatinine from 500
to 800 lb. Angus males were unusual in having a
high uric acid average, the highest glucose aver.
age and the highest urea average at 500 lb.; and
the lowest creatinine average at 800 Ib. Angus
males were also characterized by a marked drop
in creatinine from 500 to 800 lb. Angus females
were marked by a high uric acid average and the
highest hemoglobin, urea, amino acid and creati-
nine averages at 500 lb.; and the highest hemoglo-
bin, amino acid and uric acid levels at 800 lb. b.
wt. Angus females had the lowest blood glucose
values at both 500 and 800 lb. b. wt. These rela-
tionships probably have some bearing on the faet
that the Hereford males are the fastest and most
efficient gainers and that Angus females have the
lowest average rates and efficiencies of gain. (See
Oregon State College Exp. Station Tech. Bull.
No. 36).

370. Effects of diverting renal and adrenal
vein blood into portal vein on kidney fune-
tion and Goldblatt hypertension in dogs.
W. G. Kusicex anv A. P. TuHau.* Depis. of
Physical Medicine and Rehabilitation and Sur-
gery, Univ. of Minnesota, Minneapolis.

The objectives of this investigation were a)
to study the effect of diverting renal vein blood
flow into the portal vein system on renal function
and hypertension produced by Goldblatt clamps;
b) to study the effects of passing the adrenal vein
blood directly into the liver on Goldblatt hyper-
tension. Renal vein blood was diverted into the
portal vein by either a reverse Eck fistula or 4
direct renal to portal vein anastomosis. Clearance
rates of paraaminohippurate and creatinine were
used. Goldblatt clamps on the renal arteries of
4 control dogs resulted in an average blood pres-
sure elevation of 40/30 mm Hg, with a trend
toward a reduction in renal blood flow and glomer-
ular filtration. After a reverse Eck fistula in 4
dogs, average renal blood flow values indicated a
fall of approximately 20% with no change in ar-
terial blood pressure. Subsequent application of
Goldblatt clamps resulted in an elevation in
arterial blood pressure similar to the control
animals. In 2 dogs, a direct renal to portal vein
anastomosis yielded similar results. Diverting

oxy
full
was
mor
mol
of |
ven
or ¢
wit]
inte
mea
sion
flow
alws
arou
conf
venc

plume 15

1S males
sual in

at 500
e lowest
highest
usual in
>, anda
ossessed
and the
glucose
roup to
rom 500
laving a
se aver-
lb.; and
. Angus
ed drop
- females
and the
d creati-
emoglo-
00 Ib. b.
- glucose
ese rela-
the fact
nd most
have the
ain. (See
-h. Bull.

adrenal
ey fune-
in dogs.
Depts. of
and Sur-

were @)
‘in blood
function
; clamps;
enal vein
tt hyper-
into the
tula or a
Ylearance
line were
rteries of
ood pres-
a trend
d glomer-
tula in 4
dicated 4
ge in al-
cation of
vation in
e contra
yrtal vein
Diverting

March 1956

adrenal vein-blood directly into the liver produced
no obvious alterations in the Goldblatt hyperten-
sion in 2 dogs. Since none of these procedures
produced any substantial changes in the hyper-
tension, these data may be of interest in the VEM,
YDM and other theories concerning hypertension.
(Supported by PHS grant H 1539.)

31. Physiologic mechanism of hypoxemia in
atrial septal defect. Hirosu1 Kurpa,* Pau
Novack,* Fiorence W. Haynes, LEonaARD A.
Cops, JR.* AND Lewis Dexter. Depts. of Medi-
cine, Peter Bent Brigham Hosp. and Harvard
Med. School, Boston, Mass.

Hemodynamic data obtained by cardiac cathe-
terization in 60 cases of atrial septal defect have
provided clues regarding the physiologic mecha-
nism of the hypoxemia occasionally seen in this
condition. Pulmonary vein blood samples were
obtained in 8 of the 21 cases exhibiting arterial
oxygen unsaturation and in each instance was
fully saturated. Similarly, pulmonary vein blood
was fully saturated in 17 of 19 cases with pul-
monary flows exceeding 10 1/min/m?. Thus pul-
monary factors can be excluded as possible causes
of hypoxemia, confirming the explanation that a
veno-arterial shunt is responsible. The presence
or degree of unsaturation could not be correlated
with the level of right atrial pressure or with the
inter-atrial pressure relation when this could be
measured. Mild degrees of hypoxemia were occa-
sionally found in cases with a high pulmonary
fow-systemic flow ratio, but severe degrees were
ilways associated with a reduction in this ratio
wound unity. These studies, therefore, do not
confirm the generally accepted concept that the
veno-arterial shunt is the result of a reversal of
the inter-atrial pressure gradient. Rather, they
suggest that hypoxemia is the result of veno-
wterial mixing, which is facilitated when flow
tatering the communicating atria from the re-
spective venous systems approaches equality.

2. Tolerance to heat exposure of thirsting
mammal. Harotp Lamport. Lab. of Physiol-
ogy, Yale Univ. School of Medicine, New Haven,
Conn.

The thermal response of a thirsting mammal
posed in a dry environment hotter than body
mperature is estimated, assuming that the ani-
nal is covered with fur or clothing which is not
wetted by perspiration but permits its free evapo-
tution. Considering a sinusoidal diurnal fluctua-
tion in environmental temperature or a fixed one,
this analysis leads to the conclusion that the larger
the animal the better it can withstand a period of
leat exposure without water, provided that food
8 freely available. However, allowing for the
tetabolism needed to permit foraging for food

AMERICAN PHYSIOLOGICAL SOCIETY

115

where it is scarce indicates an optimum size for
the thirsting mammal under given stressful
conditions. Even under normal conditions and
with access to water, the size of an animal must
be limited by the availability of food and the re-
lated foraging distance.

373. Respiratory rate patterns of hibernation
in the 13-lined ground squirrel. BARBARA R.
LANDAU AND ALBERT R. Dawe (introduced by
W. B. Youmans). Dept. of Physiology, Univ. of
Wisconsin, Madison.

Patterns of the respiratory rate of 13-lined
ground squirrels were obtained utilizing strain
gage and thermistor recording methods. Data
were taken on unanesthetized and anesthetized
animals in a nonhibernating season and in a hi-
bernating season. In the latter case records were
made during: a) induction, 6) hibernation and
c) arousal from the hibernating state. The re-
peating pattern of respiration in deep hibernation
was seen to consist of a few breaths followed by a
long pause, minutes in duration. However, it was
found that small faster respiratory movements
occurred which are not apparent to the observer.
As arousal proceeded, these smaller movements
increased in frequency and amplitude, replaced
the large breaths, and gave rise to a hyperven-
tilative pattern. This increase in respiratory rate
paralleled very closely the change in cardiac
frequency. Some of the cardiac arrhythmias which
are characteristic of hibernation were shown to be
associated with respiratory movements.

374. Digestion and absorption of acetyl-
tryptophan. Ratpu R. LANGNER (introduced
by E. L. Smrrx). Univ. of Texas, Dental Branch,
Houston.

Acetyl-L- and acetyl-p-tryptophan were incu-
bated at 37°C in phosphate buffer at px 7.7, for
24 hr. with carboxypeptidase, 3 X crystallized.
The liberation of free tryptophan was indicated
by use of paper chromatography and L-tryptophan
was assayed with L. plantarum. Carboxypeptidase
was inactive with acetyl-p-tryptophan, and only
26% of the i isomer was hydrolyzed. Other di-
gestive enzymes were inactive with both isomers.
This data was interpreted to mean that little or
no hydrolysis occurs in the intestine before ab-
sorption. To further substantiate this interpreta-
tion, fasted rats were fed, via a stomach tube, 246
mg acetyl-L-tryptophan, and within 2 hr., a con-
siderable quantity of the compound was found in
the plasma. The gut was analyzed for acetyl-
tryptophan at varying intervals using the Cori
technique, and acetyl-L-tryptophan was absorbed
at a slower rate than free L-tryptophan. These
and other data will be presented showing the rate
of absorption of the 2 isomers of tryptophan and

116

acetyltryptophan. The differences in the absorp-
tion of these compounds in man and the rat will
be discussed.

375. Carbon dioxide retention in working
divers breathing nitrogen-oxygen mixtures
at depth. Epwarp H. Lanpuier (introduced
by Orr E. Reynoups). Exptl. Diving Unit,
Naval Gun Factory, Washington, D. C.

When the ability of divers to tolerate exposure
to increased partial pressures of oxygen was in-
vestigated under various conditions, it was found
that toxic manifestations appeared earlier when
the divers breathed a nitrogen-oxygen mixture
at depth than when they were exposed to pure
oxygen at the same partial pressure. Investigation
of possible reasons for the difference in latent
period included measurement of end-tidal pCO:
and other respiratory variables in 17 diver-sub-
jects under several different conditions of breath-
ing medium and depth during underwater swim-
ming and rest. With exposure to a 45% O.—55% N
mixture at 4 atm. (99 ft of sea water), the mean
end-tidal pCO. during swimming was 53.5 mm
Hg, and individual values as high as 72 mm Hg
were recorded. The corresponding mean value
when the subjects were close to the surface breath-
ing air was 46.7 mm Hg, and almost identical
values were obtained with oxygen at 26 feet and
with 45% 0.-55% He at 99 ft. The CO, levels
associated with the presence of nitrogen were
considered high enough to explain enhanced sus-
ceptibility to oxygen poisoning and in some cases
to produce symptoms of carbon dioxide intoxica-
tion. High end-tidal pCO2 appeared to be second-
ary to inadequate pulmonary ventilation (low
RMV), but the mechanism of this apparent res-
piratory depression is as yet unexplained. Some
divers were found to have exceptionally high
alveolar and arterial pCO2 even under normal
conditions.

376. Electrical correlates of the human uterus
in labor. 8. D. Larxs (introduced by W. A.
SELLE). Dept. of Biophysics, School of Medicine,
Univ. of California at Los Angeles, Los Angeles.
Electrical patterns associated with the uterine

contractions of labor have been recorded from

the abdominal wall and from more remote points.

The pattern shows a period equal to that of the

uterine contraction, and an amplitude in the range

4-20 mv. Variability and relatively low potentials

are usual early in labor, with a more regular pat-

tern of higher amplitude becoming evident in
well-established labor. Certain of the observed
potentials are reminiscent of the cardiac potential
in part, showing initially a spike or biphasic vari-
ation, followed by a long slow wave. These ob-
servations are similar to and in agreement with
the smooth muscle work of Bozler and others.

FEDERATION PROCEEDINGS

4 4

Volume ij

Propagation velocities have been estimated from
the biphasic variations, and are of the order of g
few millimeters per second. In instances of pito.
cin-induced labor the propagation velocity hag
been observed to be higher, and studies are being
made to investigate this aspect. Fourier analyseg
have been made of the observed waveforms, and
the calculated harmonic patterns are suggestive
of the presence of pacemaker areas and rhythms,
An electric correlate at twice frequency seems to
be present and to play a role in contraction, along
with other harmonics. The existence of 2 pace-
maker areas for the uterus simplex has been sug.
gested in the past by animal experimentation and
by embryological considerations, and may ae-
count for the variability in pattern. (Supported
by a research grant from the PHS RG 4462.)

377. Distribution of circulating eosinophils
of the rat in cold stress. NatHan LAvEnpA
AND RoscorE G. Bartiert, JR. (introduced by
JoserH L. Jounson). Howard Univ. College of
Medicine, Washington, D.C.

Seventy male Sherman rats, averaging 150 gm,
were subjected to cold stress (10°C) for 1 hr., the
animals being maintained in separate cages. Eo-
sinophil and differential determinations were
made prior to, 4 hr. and 24 hr. after exposure,
Blood was taken simultaneously from the left ven-
tricle and the tail. Before exposure to cold there
was invariably a greater concentration of eosino-
phils in the tail than in the circulation from the
heart. Four hours after exposure there was a large
rise of eosinophil concentration in the tail vein
with a moderate rise in the blood of the heart.
Twenty-four hours later the eosinophil concentra-
tion in both the heart and tail returned to the level
of the unexposed animals. (Supported in part by
contract with the Atomic Energy Commission.)

378. Method for in situ study of aortic elas-
ticity in the dog. RicHarp W. LAwTon AND
Leon C. GREENE.* Aviation Med. Acceleration
Lab., Johnsville, and Dept. of Physiology, Univ.
of Pennsylvania School of Medicine, Philadelphia.
By means of a frame-by-frame analysis of slow

motion moving pictures, the relation between

pressure, diameter and length of an abdominal
aortic segment may be determined. Small beads

are sewn in the aortic wall with fine atraumatic } |

sutures after its exposure. The pressure is meas-
ured with a capacitance manometer. The catheter
is passed via the femoral artery and its position
with relation to the beads in the aortic segment
under study is ascertained by x-ray. The oseillo-
scope trace of the pressure is reflected by means of
a front-surfaced mirror so that it appears in each
frame. Color pictures are taken with a Mitchell
camera at 120 frames/sec. with exposures of 1/250-
1/1000 sec. The natural frequency of the catheter

olume 15

ed from
‘der of g
of pito-
city has
re being
analyses
‘ms, and
ggestive
hythms,
seems to
m, along
2 pace-
een sug-
tion and
may ac-
ipported
62.)

nophils
4AVENDA
luced by
‘ollege of

150 gm,
_hr., the
ges. Ko-
ns were
xposure,
left ven-
yd there
f eosino-
from the
s a large
tail vein
1e heart.
ncentra-
the level
part by
ssion.)

tic elas-
TON AND
eleration
yy, Univ.
adel phia.
s of slow
between
»dominal
ill beads
raumatic
is meas-
catheter
position
segment
e oscillo-
means of
s in each
Mitchell
of 1/250-
catheter

March 1956

manometer System is about 60 cps, which is suffi-
ciently high for the frame rate used. Two GE 750R
lamps provide illumination. Double layers of heat-
absorbing glass prevent undue heating and drying
of the operative field. The distances between
heads on each projected frame are determined by
means of a precision cathetometer with a 5 cm
travel. Preliminary data indicate that diameter
changes at normal mean and pulse pressures are of
the order of 2% with a reading error of approxi-
mately 0.25%. The length changes are approxi-
mately 180° out of phase with the diameter
changes, which are nearly in phase with the pres-
ure. A total excursion in length of only 1% usu-
ally occurs.

319. Dietary minerals and vitamin E in mouse
‘paralysis.’ Y. Cuotuna Pun Lzs,* Josprx T.
Kine AnD Maurice B. VisscuEr. Dept. of Physi-
ology, Univ. of Minnesota, Minneapolis.

It has been reported that the mineral content
of a vitamin E-deficient diet is a determining
factor in the development of adult mouse ‘paral-
ysis’ (Proc. Soc. Exper. Biol. & Med. 88: 406, 1955).
further experiments have been carried out to test
the effect of the trace elements, Zn, Mn, Cu, Co
and I, and ferric and ferrous iron. Eight groups
of inbred C;H male mice were givén the experi-
mental diets after weaning and were housed in a
rom maintained at 76+2°F. The gain in weight
of the group on a low vitamin E diet, containing
asalt mixture with no added trace elements, was
geatly retarded. Typical symptoms of ‘paralysis’
weurred at about 8 mo. in 100% of the mice on a
low vitamin E diet made with a modified Hubbell-
Mendel-Wakeman salt mixture containing no
trace elements. A similar syndrome also developed
when the trace elements in a modified Jones-
Foster salt were removed. On the other hand,
symptoms of ‘paralysis’ did not appear when the
ttace elements were added to the modified H-M-W
alt. Substituting ferrous sulphate for ferric
phosphate in the H-M-W salt mix in a diet con-
taining adequate vitamin E raised the incidence
‘paralysis’ from zero to 100%. This was pre-
vented by adding trace elements to the salt mix.
Apparently one or more trace elements stimulated
gowth and prevented the development of ‘paral-
jsis’ in mice on a low vitamin E diet.

0. Influence of steroids on growth of Neuro-
spora crassa. GABRIEL LESTER,* Davip STONE
AnD Oscar HecutTEr. Worcester Fndn. for Expil.
Biology, Shrewsbury, Mass., and Dept. of Mi-
crobiology, Yale Univ. School of Medicine, New
Haven, Conn.

The biological roles which steroids serve in
ticroorganisms are obscure. It has long been
fnown that certain microorganisms contain siz-

AMERICAN PHYSIOLOGICAL SOCIETY

117

able amounts of sterol, principally ergosterol.
More recently, it has been established that many
microorganisms contain the enzymatic apparatus
for a variety of steroid transformations, including
specific hydroxylations, dehydrogenations, degra-
dation of the sterol side-chain, etc. These conclu-
sions are derived from the characterization of the
transformation products produced following in-
cubation of various steroids with microorganisms.
These observations raise the question as to
whether the function of these microbial enzymes
is to transform naturally occurring sterols into
active steroids which, similar to mammalian
steroid hormones, regulate cellular metabolism
in microorganisms. As one approach to this prob-
lem we have investigated the influence of mam-
malian steroid hormones and certain of their
derivatives upon the growth of Neurospora crassa.
In these experiments typical steroids of the Cis,
Ciy and Cz; series as well as cholesterol and ergos-
terol have been studied. The most striking result
observed was the finding that desoxycorticos-
terone (DOC) caused marked inhibition of
growth. Related corticosteroids had little or no
inhibitory activity. Testosterone and proges-
terone, while possessing definite inhibitory activ-
ity, had only small effects relative to DOC.
(Supported by a grant from The Commonwealth
Fund.)

381. Rotatory dispersion of DNA and its de-
rivatives. B. H. LevEpAHL AND T. W. JAMES
(introduced by T. L. Jann). Dept. of Zoology,
Univ. of California, Los Angeles.

A clear definition of the ‘native state’ of de-
soxyribose nucleic acid rests on the possibility of
establishing a physical method capable of detect-
ing irreversible configurational changes in this
molecule. Determinations of the rotatory dis-
persion of DNA before and after various denatu-
ration procedures, as well as determinations on
its nucleotide and nucleoside derivatives, have
yielded information which is highly promising
as a single physical criterion of its ‘native state.’
Determination of the rotatory dispersion for each
of the derivatives at several pH values points to
the possibility that structural interpretation may
be in order. The rotatory dispersion of these
compounds has been measured over the range
from 600 my to 320 mu. The specific rotation, the
slope, and the intercept of the ‘Drude plot’ have
been determined for each of the compounds at
several pH levels. In addition these same factors
have been determined for several mixtures of
desoxynucleotides and nucleosides.

382. Formylglycinamidine ribotide and 5-
aminoimidazole ribotide—intermediates in
purine biosynthesis. Bruce L&EvENBERG

118

AND Irvine MELNicK (introduced by R. G.
Hoskins). Div. of Biochemistry, Massachusetts
Inst. of Technologu, Cambridge.

A new arylamine compound arising as a product
of reaction between formylglycinamide ribotide
(FGAR), glutamine and ATP in the presence of
K+, Mg** and an ethanol fraction of pigeon liver
extract has been isolated and identified as 5-ami-
noimidazole ribotide (AIR). During the course
of further study of this reaction, it was noted
that the enzymatic components responsible for
the synthesis of AIR could readily be separated
by fractionation with ammonium sulphate into
2 fractions, neither of which, separately, could
carry out the conversion of FGAR to AIR. How-
ever, in the presence of FGAR, glutamine and
ATP, fraction I (which precipitated between
0 and 35% saturation of ammonium sulphate)
catalyzed the liberation of glutamic acid and the
formation of a heat-stable intermediate. This
latter substance has now been isolated in pure
form as the barium salt and has been identified
on the basis of chemical analyses, electrometric

titration and metabolic properties as (a-N-
formyl)-glycinamidine ribotide (FGAM). AIR

was formed upon incubation of fraction IT (which
precipitated between 45 and 60% saturation of
ammonium sulphate) with FGAM and ATP. The
site of action of the antibiotic, L-azaserine, in
the biosynthesis of inosinic acid has now been
confined to the reaction catalyzed by fraction I
(i.e. the synthesis of FGAM from FGAR). In this
reaction, azaserine acts as a competitive inhibitor
of glutamine.

383. Effect of estrogen and ethionine on cold-
induced fatty liver of rats. R. L. JASPER
(introduced by G. W. Moutnar). Dept. of En-
vironmental Medicine, Army Med. Research Lab.,
Fort Knox, Ky.

It has been observed that fasting female rats
rapidly accumulate liver lipides during exposure
to low temperature (1-4°C). Investigations were
undertaken in an attempt to determine a possible
relationship between the cold-induced fatty liver
and the ability of rats to resist and adapt to cold.
An increase in liver fat is apparent after 2 hr. of
exposure to cold and develops at a rate comparable
to that observed in ethionine-treated fasting rats
maintained at 25°C. Attempts to intensify this
cold-induced increase in liver fat by the injection
of ethionine prior to cold exposure gave variable
results. In some instances ethionine further in-
creased the accumulation of liver fat, but in a
number of experiments it exerted a significant
lipotropic action. Ethionine may, to a limited
extent, take part in certain reactions which in-
volve methionine in fat metabolism. In rats at
room temperature this action may interfere with

FEDERATION PROCEEDINGS

Volume 16

the efficient utilization of methionine, causing an
accumulation of liver lipid. In animals subjected
to cold, the antagonist appears to substitute for
the normal metabolite. The lipotropic effect of
ethionine in cold-exposed animals appears to be
related to the systemic estrogen level. Estrogen.
treated and untreated castrate female rats when
exposed to cold showed the same increase in liver
lipides as intact animals. However, a priming
dosage of estrogen given 4 hr. before cold exposure
resulted in a marked lipotropic action of injected
ethionine.

384. Influence of anomalous viscosity of
blood upon resistance to flow. Matraey
N. Levy. Physiology Dept., Albany Med. Col-
lege, Albany, N. Y.

In order to assess the relative importance of
vascular distensibility versus the anomalous
viscous behavior of blood, the changes in resist-
ance produced by alterations in intravascular
pressure were compared for blood and for more
homogeneous perfusates, such as saline, dextran,
and plasma. An artificial perfusion system was
employed which made it possible to vary the
absolute values of arterial and venous pressures in
an isolated dog’s leg, while maintaining a constant
arteriovenous gradient. For equivalent pressure
gradients, the flows with homogeneous perfusates
were two to four times as great as those with arte-
rial blood. In all cases, however, flow increased
progressively as the absolute levels of pressure
were elevated, indicating a definite distension of
the higher resistance vessels. Furthermore, the
percentage changes in flow were virtually identi-
cal with blood and with the other perfusates.
Therefore, no important influence upon resistance
could be ascribed to the anomalous viscosity of
blood. It has been demonstrated, therefore, that
increased intravascular pressures elicit vascular
distension and a consequent acceleration of flow.
Since increased tube radius augments, while in-
creased velocity of flow diminishes, the apparent
viscosity of blood in small vessels, it is evident
that these effects would tend to neutralize each
other under the conditions of these experiments.
This probably accounts for the resultant negligible
effect attributable to the anomalous viscosity of
blood.

385. Serum lipoprotein and_ cholesterol
changes in nephrectomized dogs main-
tained by peritoneal dialysis. Lena A.
Lewis, Wittem J. Koirr* anp Irvine H.
Pace. Research Div., Cleveland Clinic Fndn.
and the Frank E. Bunts Educational Inst., Cleve-
land, Ohio.

That the kidney is an important factor in deter-
mining serum cholesterol and lipoprotein concet-

ter

‘olume 16

vusing an
subjected
‘itute for
effect. of
ars to be
Ustrogen-
ats when
e in liver

priming
exposure
"injected

sity of
AATTHEW
Led. Col-

‘tance of
210malous
in resist-
vascular
for more
dextran,
stem was
vary the
»ssures in
constant
pressure
erfusates
vith arte-
increased
pressure
ension of
nore, the
ly identi-
rfusates.
esistance
cosity of
ore, that
vascular
a of flow.
while in-
apparent
3 evident
lize each
eriments.
negligible
scosity of

olesterol
s main-
Lena A.
vINE H,

ac Fndn.
st., Cleve-

‘in deter-
n concen-

March 1956

trations is recognized. To further assess its role
these studies on nephrectomized dogs maintained
by peritoneal dialysis were carried out. Serum
cholesterol and lipoproteins, determined by ultra-
centrifugation at d 1.21 using KBr and NaCl, were
measured before and after nephrectomy. Some
dogs were fed a high protein diet post-nephrec-
tmy, while others were not fed. Intermittent
peritoneal dialysis was carried out, a total of
approximately 3 liters of fluid being used daily.
five days post-nephrectomy the serum choles-
terol concentration had increased 28% in the
unfed animals and 48% in those receiving a high
protein diet. The lipoprotein pattern showed an
increased concentration of low density —S20-40
components, an increased amount of —S10-20
normally not present or present in very small
concentration, and a decreased concentration of
-§1-10 component, present in large amounts in
the normal. Analysis of the peritoneal dialysate
showed only trace amounts of cholesterol and
lipoproteins, so that total loss by this path was
mall. Studies made 8, 10 and 14 days after ne-
phrectomy showed somewhat greater increases
in cholesterol and lower density lipoprotein con-
centration, with no further change in —S1-10
lipoprotein concentration. These changes in
grum lipoprotein and cholesterol concentration
in the nephrectomized dog will be discussed and
compared with those occurring following kidney
damage.

6. Binding of sodium and potassium to
muscle proteins. Marc 8. Lewis AND HARRY
A, Sarorr (introduced by S. 8. Spicer). Natl.
Inst. of Arthritis and Metabolic Diseases, NIH,
Bethesda, Md.

The binding of sodium and potassium to myosin
\, myosin B, actin, and bovine and human serum
albumin was determined by measuring the activi-
ties of these ions in the presence of the proteins
ising cation permeable permselective membranes
as specific electrodes. The dependence of the ex-
tent of binding of potassium and sodium ions to
myosin A and B upon the metallic and hydrogen
im concentrations suggests that the imidazole
groups of histidine and the e-amino groups of
lysine are involved in the binding of these metallic
ins. Because the sodium and potassium ions
compete with hydrogen ions for these sites, the
calculated values of the thermodynamic constants
for these interactions are dependent upon the
values assumed for the pk’s of these groups.
Myosin B was found to bind less potassium than
myosin A, but this may be due to fewer sites
being available rather than to lower association
constants. It was found that actin and bovine
ad human serum albumin did not bind potassium.
The addition of actin to myosin A reduced the

AMERICAN PHYSIOLOGICAL SOCIETY

119

amount of potassium bound, possibly by blocking
some of the sites which would otherwise be avail-
able to potassium ions. This may also be responsi-
ble for the less extensive binding of potassium to
myosin B than to myosin A.

387. Gradients of motor function in the whole
cerebral cortex of the unanesthetized
monkey. Joun C. Litty, Joun R. Hucues,*
AND THELMA W. Gatkin.* Natl. Inst. of Mental
Health, Bethesda, Md.

A study of the literature on motor mapping of
the anesthetized cerebral cortex of the monkey
shows that electrical stimulation of a large frac-
tion of the cortex can produce movements
(ScHAFER, W. K. Smitu, Livineston, WALKER
AND WEAVER, VON BECHTEREV). The extent of
the responsive areas varies with anesthetic level;
the area most resistant to anesthesia is the classi-
cal precentral ‘motor area.’ In each of 6 monkeys,
we implanted one or two 1-cm? arrays containing
25-121 electrodes over various cortical areas,
from occipital to frontal. In the unanesthetized
state, motor responses were elicited from every
such area. In order to see if there were any ‘silent
gaps’ in the motor map, we stimulated cortex at
each of 610 electrodes implanted over a large
fraction of a single hemisphere in one monkey.
Movements were elicitable at each electrode, from
frontal pole to those in area 17, and from near the
midline to those over temporal cortex. No gaps
were found. These results confirm those of the
above workers: in areas 6 and 8 (eyes and head);
4, 8,1, 2, § and 7 (face, arm, back, and leg); 19,
18, and 17 (eyes and head); and 2/ and 22 (ear).
In addition, eyes, head, and ear movements were
found in area 9 all the way to the frontal pole;
and eye-head movements in the lateral part of
22. Threshold values are lowest in areas adjacent
to central sulcus, and increase with distance ante-
riorly and posteriorly from this region. These
results suggest that motor functions are dis-
tributed throughout the unanesthetized cerebral
cortex in the normal state, and, imply, when
correlated with sensory maps, that each small
area of cortex is truly ‘sensori-motor’ with a pre-
ponderance of one or the other function.

388. Physiological properties of cerebral
cortical motor systems of unanesthetized
monkey. JoHN C. Litiy, Joun R. Hucues*
AND THELMA W. Gatxin.* Nail. Inst. of Mental
Health, Bethesda, Md.

In 7 unanesthetized monkeys containing arrays
of 25 to 610 electrodes in various cortical areas,
studies of motor responses have been made in
various physiological states. The classical thresh-
old motor response is elicited by trains of electri-
cal pulse-pairs occurring at 60/sec. in 3- to 5-sec.

120

trains repeated once per minute. The critical fre-
quency range for this response is from about 30
to 250 pulses/sec. During a threshold train of
indefinite length, a response occurs only once,
after a latency of #4 sec. and lasts 10-15 sec. In
a long series of trains with a given train duration,
there is a minimum critical value of inter-train
interval below which only one response occurs.
Train durations less than a critical value do not
produce responses unless current is increased
above the threshold value for the train of indefi-
nite duration. The threshold values rise during
dozing and sleep, fall after meals, and can either
rise or fall during contemporaneous motor activity
not elicited by the electric stimuli. Very low fre-
quency (1-2/sec.) stimuli produce responses which
show similar changes in threshold, can be sup-
pressed and/or facilitated by previous high
frequency subthreshold trains, and show ‘spon-
taneous’ changes in amplitude varying continu-
ously, but not necessarily periodically, over 5-
10 sec. intervals. Using pulse pairs of opposite
sign and of equal charge, of various shapes and
various inter-pulse intervals, it was found that
the constant-quantity interval for excitation of
these two types of responses is approximately
100 pu sec.

389. Absorption of dietary cholesterol in man.
T. M. Lin, Esko Karvinen anp A. C. Ivy
(introduced by E. Roopa Grant). Dept. of
Clin. Science, Univ. of Illinois, College of Medi-
cine, Chicago.

A study has been made of the extent of the fecal
elimination of sterols by 9 young healthy subjects
when fed a basal relatively low fat and low choles-
terol diet to which after a control period increas-
ing amounts of cholesterol were added weekly.
The basal diet for the period of 1 wk. provided
approximately 20 gm of fat, 120 gm of protein,
and 490 gm of carbohydrate daily. It was adequate
in reg4rd to vitamins, bulk and minerals. The
daily menu yielded approximately 2600 calories
on the average. Feces were collected from the
4th-7th day of each week of the basal diet and of
the basal diet with the added cholesterol. The
blood serum cholesterol was determined on the
7th day. The mean daily total sterol elimination
on the low fat and low cholesterol basal diet was
908 mg; when 1 gm of cholesterol was added,
1341 mg; when 3 gm of cholesterol were added,
2720 mg; when 6 gm of cholesterol were added,
4900 mg. When corrected for the basal sterol
elimination, the percentage of the apparent choles-
terol absorption was 56.7% (0.6 gm) for 1 gm of
added cholesterol; 39.6% (1.2 gm) for 2 gm; and
33.5% (2.0 gm) for 6 gm. When 6 gm of cholesterol
was added to the diet, the maximum individual
extra sterol elimination amounted to 81% and

FEDERATION PROCEEDINGS

Volume i

the minimum to 52%. The blood cholestero|
values remained constant during all 4 dietary
regimens.

390. Use of radioactive carbon compounds
for studying nutritional requirements of
Daphnia magna. V. F. LINDEMAN AND LERoy
Davis.* Dept. of Zoology, Syracuse Univ., Syra-
cuse, N. Y.

It can be demonstrated by the use of radioactiye
carbon compounds that Daphnia magna cap
utilize dissolved materials from the media in
which it lives. Approximately 25 mature animals
kept relatively free of microorganisms with anti-
biotics, were incubated in Warburg flasks for
12 hr. at 25°C in 3 ml of artificial pond water con.
taining .01 mg/ml of one of the following com-
pounds: sodium butyrate 1-C'*, sodium acetate
1-C, glycine 1-C4, glutamic acid 1-C", sodium
formate 1-C4 and glucose U.L. On the assumption
that utilization of a compound results in either
oxidation or decarboxylation and the subsequent
production of CO, the carbon dioxide produced
in each flask was collected in saturated NaOH,
precipitated as BaCl;, dried, weighed and meas-
ured in a flow counter. The radioactivity of the
animals was also measured after careful washing
and drying. The table shows the number of micro-
grams of compound recovered as CO per mg
dry weight of animal for 12 hr., and the number
of micrograms recovered in the animals.

Compound No. Exp. as C“Oz2 In Animals Total
Sodium acetate 6 .622 -495 1.122
Sodium butyrate 7 483 .143 0.620
Glycine 6 .150 .080 0.230
Glutamic acid 6 265 .236 = 0.501
Sodium formate 6 .035 .015 0.050
Glucose 6 .130 .098 0.228

(Supported by a Natl. Inst. of Health Grant
No. G-3947.)

391. Further evidence for the fixed charge
hypothesis. GiLBperT Linc AND ARLENE
ScHMOLINSKE.* Psychiatry Div., Univ. of Illi-
nois, Chicago.

By utilizing the 2-pi geometry and the high
gamma efficiency of a scintillation well counter it
became possible to measure continually the total
amount of an ion tagged with a high energy gamma
emitting isotope in a single muscle fiber or a0
isolated small bundle of fibers while gaining or
losing such ions. Uniquely this method enables
one to follow accurately the uptake or loss of
tagged ions in the initial seconds of their exchange.
Thus it became possible clearly to differentiate
between the rapid loss of Nat from the extra-
cellular space of a muscle and early fast efflux of

7olume if

rolesterol
t dietary

npounds
nents of
'D LeRoy
tv., Syra-

dioactive
gna can
media in
> animals
vith anti-
lasks for
ater con-
ing com-
1 acetate
‘, sodium
sumption
in either
bsequent
produced
1 NaOH,
nd meas-
ty of the
washing
of micro-
» per mg
> number

h Grant

charge
ARLENE
. of Illi-

the high
ounter it
the total
y gamma
er or al
sining oF
| enables
r loss of
xchange.
erentiate
le extra-
efflux of

March 1956

the intracellular phase. Such studies showed that
not all Nat were equally exchangeable. This is
in agreement with the idea that since most of the
intracellular fixed anionic charges are occupied
by K*, only a part of the less strongly adsorbed
Nat could occupy such sites, and these would
make up the comparatively small less-easily ex-
changeable fraction under normal conditions.
Other Na* not occupying sites would exist at
varying energy levels and, depending also on their
instantaneous locations, of different exchange-
ability. It was further shown that if the intra-
cellular K* concentration was lowered by previous
or simultaneous treatment of the cells with a
low-K* Ringer solution, the outward flux rate
of Nat sharply decreased, a phenomenon already
reported by Keynes. In our opinion this could be
explained by an increase in the number of anionic
sites now evacuated by K* and available to Nat,
hence an increase in the less easily exchangeable
fraction. In the course of 9 days muscles cells
continually gained Cs* from a Ringer solution
containing an extra 2.5 mM of tagged Cs*. Again
the total time course appeared non-Fickian,
further supporting the fixed charge hypothesis.
(Aided by a grant from the Muscular Dystrophy
Assn. of America.)

392. Effect of anemic rabbit plasma on ery-
thropoiesis in the rat. W. H. LINKENHEIMER,*
W. C. Grant anv H. Bercer.* Research Div.,
American Cyanamid Co., Lederle Labs., Pearl
River, N. Y.

Several investigators have suggested that ery-
thropoiesis is under hormonal regulation. It has
been demonstrated that injections of plasma from
anemic animals into normal animals are followed
by a reticulocytosis and an increase in hematocrit
%. This paper presents a demonstration of this
activity by an entirely different method. This
laboratory recently reported an increase in the
P® uptake by the deoxynucleic acid of the rat
spleen after hemorrhage or anoxia, two conditions
known to stimulate erythropoiesis. It was, there-
fore, of interest to determine whether plasma from
anemic animals would also increase the P* up-
take. Plasma obtained from rabbits made anemic
by injections of phenylhydrazine was adminis-
tered to normal rats. This was followed by a tracer
dose of P32. The animals were later killed and the
spleens excised. The spleens of 3 or 4 rats were
pooled and the DNA fraction was isolated. The
final values were expressed as cpm/0.1 mg total P.
Spleens of rats injected with plasma from anemic
tabbits had a P%? uptake of 2-4 times that of
control animals injected with normal rabbit
plasma or saline. The activity present in anemic
tabbit plasma was found to settle during ultra-
centrifugation.

eer?

AMERICAN PHYSIOLOGICAL SOCIETY

121

393. Pyruvate phosphokinase system as a
relaxing factor in glycerinated muscle.
L. Loranp anv C. Moos.* Chemistry Dept.,
Northwestern Univ., Evanston, IIl.
Identification of the adenosine triphosphate

(ATP)-creatine transphosphorylase system as 2

relaxing factor in glycerinated muscle (GooDALL

AND ANDREW SzENT-GyORGYI, AND LORAND) sug-

gested that re-phosphorylation of specially lo-

cated adenosine diphosphate (ADP) molecules
produced during contraction is the significant
step underlying relaxation. Bendall’s demonstra-
tion that myokinase, too, is a relaxing factor
supported this view. Altogether, there may be
several physiological channels producing relaxa-
tion by linking various phases of the energy
metabolism to the activity cycle of muscle through
re-phosphorylation of ADP. Lorand and Andrew

Szent-Gyérgyi suggested that the pyruvate

phosphokinase (PPK) system could serve as

another such mechanism. We now find that
glycerol-preserved rabbit psoas (prepared accord-
ing to Szent-Gyérgyi), after isometric contrac-

tion with ATP (4 mm), relaxes upon adding 10 mm

phosphoenolpyruvate (PEP) (synthesized accord-

ing to Baer) in a solution of KCl (0.1 m) and

MgCl, (4 mo) near neutrality. This relaxation is

completely reversed by adding 0.5. mm CaCle. The

extent and speed of relaxation increase with

MgCl, concentration from 1 mm to about 4 mm,

which is apparently optimal. Since added PPK

enzyme is not required for relaxation, this enzyme
was thought to be tenaciously bound by the fibers.

Indeed, homogenates of extensively washed fibers

displayed high PPK activity as shown by pyru-

vate liberation in the presence of PEP and ADP,
according to the method of Kachmar and Boyer.

Even fibers apparently free of relaxing ability

attributable to myokinase still relax upon addi-

tion of PEP. (Aided by a grant from the Muscular

Dystrophy Associations of America, Inc.)

394. Effect of phlorizin on oxidative metab-
olism in kidney. Witu1am D. LotTsPEeIcu AND
Danret M. Keuuer.* Dept. of Physiology, Col-
lege of Medicine, Univ. of Cincinnati, Cincin-
natt, Ohio.

In a previous report from this laboratory it
was shown that phlorizin in concentrations as
low as 5 X 10-‘m inhibits the oxidation of all the
substrates of the tricarboxylic acid cycle in
washed homogenates of guinea pig kidney cortex.
The general nature of this inhibitory effect sug-
gested that phlorizin acts primarily at some site
in the final common pathway of biological oxida-
tion, perhaps in the electron transport system.
In the course of experiments designed to look for
such a site, it was found that when this system
was inhibited with cyanide and an alternate elec-

122

tron acceptor (methylene blue) was added, phlor-
izin had no inhibitory effect on oxidation. Further-
more, excess adenosine triphosphate (ATP)
prevented the phlorizin inhibition in the system
without cyanide. Excess adenosine diphosphate
(ADP) in equimolar amounts was even more
effective in this respect. Adenylic acid (AMP),
on the other hand, while supporting maximal
oxidation in the control preparation, was com-
pletely ineffective in preventing the phlorizin
inhibition. If phlorizin inhibits the phosphoryla-
tion of ADP then the addition of excess ADP
(or ATP) would be expected to overcome the
inhibition, while AMP would not since it does not
ordinarily act as a phosphate acceptor. These
findings indicate that phlorizin inhibits oxidative
metabolism in the kidney at the site in the electron
transport system where ADP is phosphorylated
to ATP during the transfer of electrons from
reduced pyridine nucleotide to molecular oxygen.
Such interference with the synthesis of high
energy phosphate would certainly inhibit tubular
transport requiring such an energy source.

395. Squalene turnover in relation to choles-
terol biosynthesis. ALDEN V. Loup (intro-
duced by Nancy L. R. Bucuer). Med. Labs.,
Huntington Memorial Hosp. of Harvard Univ.,
Massachusetts Genl. Hosp., Boston.

Studies of the turnover of hepatic squalene in
the rat were undertaken to elucidate the role of
the relatively large tissue pool of squalene in the
intermediary stages of cholesterol biosynthesis.
The concentration of squalene was determined by
isotope dilution technique to be approximately
50 uwg/gm of fresh tissue. In short-term experi-
ments the total radioactivity in hepatic squalene
and cholesterol was measured at intervals up to
30 min. following the administration of a tracer
dose of C14-acetate. The resulting time curves
for these two substances were characteristic of a
precursor and its end-product. Furthermore, the
sharp initial rise in squalene activity and its steep
fall after the exhaustion of the labeled acetate
indicated that this intermediate pool has a very
rapid turnover rate. In a long-term experiment,
carrier-free tritium-labeled squalene, character-
ized by formation of the crystalline hexahydro-
chloride, was isolated from the pooled livers of
22 rats maintained for 3 weeks with tritiated body
water of constant specific activity. The hydrogen
atoms in this squalene were found to have a spe-
cific activity only 3 that of the hydrogen atoms in
the cholesterol from these livers, implying that
the total hepatic squalene turns over more slowly
than cholesterol. These data are reconciled by the
conclusion that the squalene of rat liver is sepa-
rable metabolically into a small active component
(possibly enzyme-bound) and a larger relatively
inert pool.

FEDERATION PROCEEDINGS

Volume i

396. Thyroid activity of inbred strains of
mice. JoHN B. Lyon, JR. (introduced by H. D,
BrunER). Dept. of Biochemistry, Emory Univ,
Emory University, Ga.

A series of studies are in progress to determine
hormonal differences among A, C57, and I strain
mice which exhibit gross metabolic differences,
Since these strains show different growth rates,
estimations of their thyroid activity were made
utilizing radioactive sodium iodide. The iodide
was given intraperitoneally to fed, 3-mo. old mice
in doses of 1 we. At intervals of 2, 4, 12, 24 and
48 hr. after dosage the thyroid glands were re-
moved under Nembutal anesthesia and the amount
of radioactivity was estimated with a well-type
scintillation counter. The percentage uptake as
calculated from an internal standard was found
to be approximately 14-2 times greater among A
strain mice than among I strain mice at all time
intervals. Values for mice of the C57 strain were
in general intermediate between the A and I
strains and were not significantly different from
either one. However, 1 1u of TSH given 1 hr,
prior to the iodide evoked increases in the [#
uptake of A strain mice thyroid glands that were
4 times greater than the increases evoked among
C57 strain mice. (Work done during the tenure of
a Postdoctoral Fellowship from the Life Insurance
Med. Research Fund.)

397. Demonstration of a toxin in urine of
psychotic patients. Davip I. Macut ann
Dorotuy KreMEN.* Pharmacological Div., Labs.
Sinai Hosp., Baltimore, Md.

In 1950 Macht reported a 10-yr. study of blood
from psychiatric patients by phytophysiological
methods, finding that blood sera, 1%, and spinal
fluid, 2%, inhibited root growth of Lupinus albus
seedlings in Shive’s hydroponic solutions under
controlled ecological conditions (South. M. J.).
The degree of toxicity varied with the severity of
the disease. The present research reveals the pres-
ence of such ‘toxin’ also in psychotic urine. All
urines, both in health and disease, are more
poisonous for seedling growth than blood serums
because of urea which retards root growth. On
eliminating urea with urease the specimen be-
comes less toxic. Thus a ‘toxin’ was demonstrated
in the urine of all cancer cases (J. Urol. Oct.,
1955). By similar techniques the urine of psy-
chotics was found markedly inhibitory to the
growth of Lupinus seedlings. The toxic constitu-
ents in psychoses are different from cancer ‘toxin.’
‘Cancerotoxin’ inhibits growth only of vernalized
plants and is unaffected by treatment with corti-
sone or x-rays. Psychotic urines are greatly de-
toxified in vitro by cortisone, 1:2,000,000, or by
irradiation with x-rays passed through a com-
posite filter. Boiling weakens the toxicity of psy-
chotic urine but not of cancer urine. Cancer urine

siu
fol

plume If

ains of
y H.D,
y Univ,

termine
I strain
erences,
h rates,
re. made
> iodide
1d mice
24 and
vere Te-
amount
ell-type
take as
s found
mong A
all time
in were

and |
nt from
n 1 hr,
the [#
at were
| among
snure of
surance

‘ine of
IT AND
»., Labs,

f blood
ological
1 spinal
us albus
3 under
M. J.),
erity of
he pres-
ine. All
e more
serums
vth. On
nen be-
strated
l. Oct.,
of psy-
to the
onstitu-
‘toxin.’
alized
h corti-
itly de-
, or by
a com-
of psy-
er urine

March 1956

is fatal to“mice on injection; psychotic urine is
not. The present findings with psychotic sera and
urine are useful in establishing a psychosomatic
basis for diagnosis, and as an aid in evaluating
therapeutic procedures, especially the effect of
newer ‘miracle drugs’ employed in psychiatry.

398. Continuously recording quantitative
estimation of circulatory changes in the
liver and small intestine of the dog follow-
ing Escherichia coli endotoxin administra-
tion. L. D. MacLean,* M. H. Weit,* R. J.
StisH,* W. W. Spink anp M. B. VisscHEr.
Depts. of Physiology, Surgery and Medicine,
Univ. of Minnesota, Minneapolis.

The intravenous injection of histamine-free
E. coli endotoxin 5 mg/kg into the dog results in
a precipitous fall in blood pressure and simul-
taneous elevation of portal vein pressure. A par-
tial return of systemic blood pressure occurs with
return of portal pressure to normal. Subsequently,
a further decline in blood pressure is observed.
It has been possible to prevent the initial shock
phase by excluding the liver from the circulation,
either by evisceration or by Eck fistula with
hepatic artery ligation. A device for estimating
in vivo the quantity of blood or blood products in
the liver and small intestine during the early and
late shock phases has been utilized. A phosphor-
bronze strip 3” long, 0.5” wide and from 0.01-
0.05” thick is suspended from a rigid support
which in turn supports a brass beam at its mid-
position. Strain gages of the following character-
istics are glued to each side of the phosphor-bronze
strip: Type A-1, resistance in ohms 120.8+.2
and gage factor 2.041%. The organ to be weighed
is supported from one end of the balance beam
and counterpoise added to the opposite end until
an equilibrium position is achieved. A Sanborn
polyviso recorder was used to record pressure and
weight changes continuously. A change in organ
weight causes a deflection of the beam and an
alteration in resistance in the strain gage ele-
ments which is recorded and is a linear function.
In 12 dogs a large increase in liver weight occurred
during the initial shock phase, while an increase
in weight of the small intestine was noted during
the late shock phase.

399. Actions of exogenous and endogenous
histamine on cellular potassium. WILLIAM
H. Macmiuuan (introduced by Roserr L.
Driver). Dept. of Pharmacology, Univ. of Ver-
mont, College of Medicine, Burlington.
Histamine produces not only changes in capil-

lary permeability, but also alterations of the

permeability of cellular membranes. The latter is
evidenced by a release of large amounts of potas-
sium from the tissues to the extracellular fluid
following the injection of histamine into the

: ase tae

AMERICAN PHYSIOLOGICAL SOCIETY

123

animal. The ability of three potent antihistaminic
agents (diphenhydramine, promethazine, and
pyrilamine) in blocking the kyperpotassemia in-
duced by exogenous histamine has been assessed
utilizing the intact cat. The latter two agents were
significantly effective in reducing, although not
blocking completely, the release of potassium.
It has further been established that the quantity
of potassium released by histamine liberating
substances is reduced by the antihistaminic
agents. These observations suggest a marked
similarity between the actions of exogenous and
endogenous histamine upon the transport of
potassium across the cell membrane. (Supported
by USAF-SAM Contract AF 19(604)-1093 and
PHS Grant #*B 729.)

400. Microchemical determination of alkaline
and acid phosphatase in the human stom-
ach. Rosert J. MappEen, ANTHONY L. PIETRO-
LUONGO AND ANTHONY V. Torre (introduced
by J. Eart Tuomas). V.A. Hospital, Phil-
adelphia, Pa.

These 2 enzymes were measured by the method
of Lowry (J. Biol. Chem. 207: 19, 1954) with the
exception that frozen sections were cut for chemi-
cal analysis and also mounted for purposes of
histopathology on Eastman film by the method
of Bush (Am. J. Path. 28: 863, 1952). Overall pre-
liminary results on the gastric mucosa of stomachs
obtained at autopsy, resected specimens of gastric
and duodenal ulcer and gastric cancer, and graded
for gastritis according to Hebbel (Am. J. Path.
19: 43, 1943) showed a definite and progressive
increase of alkaline phosphatase activity with
increasing severity of gastritis in the body of the
stomach which was statistically highly significant.
On the other hand, in the antrum there was only a
smail upward tendency in alkaline phosphatase
activity with increasing gastritis. Acid phospha-
tase activity also showed only a small upward
tendency in both body and antrum with increas-
ing severity of gastritis. Further studies have
shown that one of the criteria for gastritis, namely
that of intestinalization, is associated with a very
profound increase of alkaline phosphatase activity
per se over and above that produced by the other
criteria of gastritis. Further studies are in progress
to examine in detail the effect that distance from
the lesion has on both acid and alkaline phospha-
tase activity as well as the degree of gastritis. A
small series has already shown that for alkaline
phosphatase in both antrum and body there is a
progressive increase of both gastritis and enzyme
activity as the lesion is approached.

401. Retractin: Clot retraction promoting
factor in platelets and tissues. S. I. Maaa-
LINI* aND M. Sreranini. Saint Elizabeth’s
Hospital; Tufts Univ. Med. School, Boston, Mass.

124

A clot retraction promoting factor was obtained
from human platelets and tissues. Platelets were
obtained by multiple centrifugation method from
normal and polycythemic blood. Platelets were
washed 3 times with saline and used fresh or after
lyophilization and storage. Tissues were freed of
blood and homogenized by standard technique.
Active preparation was obtained by 1 of the
following procedures: 1) extraction with 75%
acetone/water mixture; 2) extraction with 65%
ethyl ether/water mixture; 3) precipitation from
ethyl ether extract by cooling at —20°C. Extracts
were free of platelets, platelet ghosts, cellular
elements and debris. After drying and resuspen-
sion in saline, these products induced good retrac-
tion of platelet-poor native human plasma and of
bovine fibrinogen solution (300 mg%) clotted by
purified bovine thrombin (10 NIH units). Power-
ful extracts, active in absence of calcium, were
obtained from human platelets and brain; weak
extracts from liver and spleen. The definition of
‘retractin’ is proposed for the newly discovered
factor. Acetone preparations of retractin con-
tained a thromboplastic factor, eliminated by
heating at 56°C/2 hr. and storage at —20°C/
48 hr. Ethyl ether preparations were free of ac-
tivity in a coagulation system in vitro. Retractin
was stable at 56°C/2 hr. and indefinitely at —20°C.
Preliminary investigation showed that this sub-
stance could be separated from other factors
present in or carried by platelets, including 5-hy-
droxytryptamine. It could represent a lipoid
substance. Clot retraction has been attributed to
presence of intact platelets. Our results, however,
indicate that an independent platelet factor may
contribute to this phenomenon. (Supported by
grants-in-aid from the PHS and American Heart
Assoc.)

402. Essential amino acids as stimulants of
pancreatic secretion. D. F. MaGgzE anp S. S.
Honé.* Dept. of Pharmacology, School of Medi-
cine, Univ. of Washington, Seattle.

Thirteen pancreatic fistula dogs have been pre-
pared. It has been found possible to make 24 hr.
collections from these over periods of several
months. Each of the animals was fed daily a fixed
amount of a proprietary dog food. To this, during
experimental periods, was added the amino acid
or other food stuff under investigation. It has
been found that additions of dl-methionine, dl
isoleucine or dl-phenylalanine amounting to
2.5 gm. daily will increase volume, proteolytic
activity, lipase and amylase each by an approxi-
mately equal amount (50%) in the case of methio-
nine and phenylalanine. Isoleucine had a smaller
effect on volume than the other 2 amino acids, a
32% increase as opposed to a 58% increase. It
also had a smaller effect on proteolytic activity

FEDERATION PROCEEDINGS

Volume i

but a greater effect on amylase than was the cage
with the other acids. The increase occurred rapidly
and with continuous amino acid feeding reached
a maximum after 3 days and then declined. This
decline was least evident in the case of phenylala.
nine. Small amounts of these amino acids which
would not produce an increase in either volume or
enzymes, when mixed with the diet, would do g9
if they were fed in solution 1 hr. before feeding, A
2% solution of dl-methionine when placed in 4
duodenal Thiry Vella loop in a pancreatic fistula
dog increased volume and enzymes.

403. Eating and drinking responses elicited
by diencephalic stimulation in unanes.
thetized rats. FrepERICK W. Marre (intro-
duced by H. D. Parton). Dept. of Physiology
and Biophysics, Univ. of Washington School of
Medicine, Seattle.

Previously we described acute hyperactivity in
unanesthetized rats induced by electrical stimu-
lation of the caudal hypothalamus through im-
planted electrodes. In an attempt to delimit the
effective hypothalamic region, stimulating elee-
trodes were placed in various diencephalic strue-
tures. Two electrode types, made with 5-mil wire,
were used. One consisted of 10 to 14 such wires
permitting multiple exploration; the other was an
adjustable bipolar electrode permitting multiple
exploration in depth. While stimulation in the
mamillary body did not produce running, it
caused a variety of other behavioral changes,
the most striking of which were eating and drink-
ing. The responses were stimulus bound, i.e. they
began promptly after the onset of stimulation
(1-20 sec.) and ceased at the end of stimulation.
Stimulation of a particular effective area usually
resulted in one type of response, eating or drink-
ing, although mixed responses were occasionally
noted. Responses were elicitable many times daily
and sometimes for periods of several days. The
short latencies of the responses seem to preclude a
humoral mechanism. Stimulation near the ante-
rior thalamus also caused eating and drinking
responses, but the latency often was longer (I-
5 min.) and the responses persisted for several
minutes following cessation of stimulation. These
findings are in agreement with the known anatomi-
cal relations between the mammillary bodies and
anterior thalamus. (Aided by a contract (NRII+-
150) between the Office of Naval Research, Dept.
of the Navy, and the Univ. of Washington.).

404. Effect of oxygen poisoning on gastrula-
tion of frog embryos. SasHa MA.ameEp (in-
troduced by Roserts Rueu). Dept. of Zoology,
Coiumbia Univ., New York City.

Nelsen has shown that freshly fertilized Rana
pipiens eggs put under oxygen pressure develop

plume 15

the cage
Tapidly
reached
2d. This
enylala-
s which
lume or
id do g0
ding. A
ed ina
¢ fistula

elicited
nanes-

(intro-
ysiology
chool of

ivity in
| stimu-
igh im-
mit the
ag elec-
c struc-
1il wire,
h wires
"was an
nultiple
in the
ling, it
hanges,
1 drink-
.e. they
1ulation
ulation.
usually
r drink-
sionally
es daily
ys. The
aclude a
ie ante-
lrinking
ger (I-
several
1. These
natomi-
lies and
NRII+
1, Dept.
n.).

strula-
ED (in-

Zoology,

>d Rana
develop

March 1956

into normal late blastulae, but, depending on the
dosage, gastrulation fails to occur or is abnormal.
The treatment was with 45 psi of oxygen added
to air at standard pressure, generally for 24 hr.;
it ended during late cleavage. Subsequently, the
present author reported that shaking reduced
the dosage time, but that less than 8 hr. treatment
was ineffective. Twelve hours’ treatment pre-
vented normal gastrulation in 100% of the em-
bryos. The following is now added: 16 hr. treat-
ment prevents gastrulation completely in 100%
of the embryos. Temperature has little if any
(net) effect on oxygen poisoning due to several
effects which are probably largely compensatory.
Lengthening the treatment makes it effective
with lower partial pressures of oxygen; thus,
even at atmospheric pressure, gastrulation is
blocked by pure oxygen. Therefore, oxygen poison-
ing is not a pressure effect and this is confirmed
by normal gastrulation after treatment with
hyperatmospheric pressures of nitrogen and even
after oxygen pressure treatment without shaking
for a duration long enough to be effective with
shaking at the same pressure. The role of pressure
then is indirect, affecting the egg medium’s oxygen
concentration (Dalton’s Law); the latter is the
effective agent. In contrast to other agents per-
mitting development only to gastrulation, oxygen
affects developmental rate little, if at all. There-
fore, at gastrulation a qualitative change must
occur bringing into play an oxygen-sensitive
system on which development is newly dependent.

405. Effect of inhibitors of oxidative phos-
phorylation on the renal transport of in-
organic phosphate. RicHarp L. MaAtviIn
(introduced by Gustav Eckstein). Dept. of
Physiology, Univ. of Cincinnati College of Medi-
cine, Cincinnati, Ohio.

The present investigation was undertaken to
determine the cellular mechanisms involved in
the renal transport of inorganic phosphate. Com-
parisons were made between the ability of guinea
pig kidney homogenates to esterify inorganic
phosphate in vitro and the ability of phosphate-
loaded dogs to maximally reabsorb filtered phos-
phate in vivo (Tm phosphate). The results of such
experiments have shown that various substances
which inhibit esterification of inorganic phosphate
in vitro also depress Tm phosphate when infused
into dogs. Of particular interest is sodium bicar-
bonate. In a previous communication it was re-
ported that the bicarbonate ion depressed Tm
phosphate in dogs. Thus, it was interesting to
note that the presence of bicarbonate in kidney
homogenates reduced their ability to esterify
inorganic phosphate, independently of the slight
change in pH of the medium. Some other sub-
stances studied were: 2,4-dinitrophenol, sodium

AMERICAN PHYSIOLOGICAL SOCIETY

125

acetoacetate, sodium malonate. Kidney homoge-
nates incubated in the presence of any of these
substances exhibit a marked reduction in the rate
of disappearance of inorganic phosphate from the
medium. If they are infused into phosphate-
loaded dogs, they all depress Tm phosphate. The
results seem to indicate that at some point in
its renal transport phosphate must be esterified,
as evidenced by the fact that certain agents which
uncouple oxidative phosphorylation, or which in-
hibit phosphorylation, also depress the rate of
renal transport of inorganic phosphate.

406. X-ray and ultraviolet effects in human
leucocytes in vitro. S. P. Maroney, Jr.
(introduced by K. M. Wiisur). Dept. of Zool-
ogy, Duke Univ., Durham, N. C.

The action of ultraviolet and x-ray on ameboid
movement and phagocytosis has been studied in
washed human leucocytes suspended in phosphate
buffer at pu 7.4. Cell motility and phagocytic
activity decreased as a function of UV dose. At
2 X 10° ergs/mm?, cell motility was 42% in the
dark and 68% when exposed to visible light indi-
cating photoreactivation. There was no difference
in phagocytic activity between cells in light and
dark. Reduced glutathione, 3 X 10-*m, provided
significant protection against the effects of UV on
cell motility whether added before or immediately
after UV but was without protective effect for
phagocytic activity. Cell motility decreased as a
function of x-ray dose to 30% at 25,800 r. When
irradiated with the same dose in the presence of
10° m glutathione, cell motility was 583%. The
addition of serum following UV or x-ray or the
x-irradiation of cells in serum caused a greater
inhibition of cell motility than when buffer alone
was used. The present experiments indicate that
human leucocytes provide favorable material for
the investigation of physical and chemical modi-
fications of irradiation effects. (Supported by the
Atomic Energy Commission.)

407. Water compartments in a cephalopod
molluse. ArTtHUR W. Martin, FLORENCE M.
HarRISON* AND JAMES D. Rosertson.* Zoology
Depts., Univ. of Washington, Seattle, and Univ.
of Glasgow, Scotland.

The phylum Mollusca includes animals of
diverse body form, size and metabolic activity.
The volume of circulating fluid has been deter-
mined for very few representatives of this phylum.
It is an interesting fact that individuals of the
molluscan class, Cephalopoda, characterized by
the greatest motility and a high continuous level
of activity, are the only molluscs reported to have
developed an entirely closed circulatory system.
In analogy with the closed system of the verte-
brates, arteries, capillaries and veins may be

126

observed. In order to determine if, as a result of
these morphological similarities, there is a dis-
tribution of blood and tissue fluid similar to that
found in the vertebrates, a series of experiments
were performed on the common large octopus of
Puget Sound, probably Octopus hongkongensis.
Blood volume determinations were done with
either colloidal HgS or T-1824, usually simul-
taneously with an extracellular fluid volume de-
termination based on the distribution of inulin or
raffinose. The blood volume ranged from 4.33 to
7.61% of the body weight with a mean of 5.83%
in a series of 13 animals. The extracellular fluid
(including the blood) ranged from 18.7 to 40.4%
of the body weight with a mean of 28% in a series
of 8 animals. It may be seen that the extracellular
fluid volume is a little higher than that reported
for vertebrates and that there is considerable
variability in the size of this fluid compartment.
(Supported in part by ONR contract N8-52011.)

408. Fatty acid synthesis from dietary carbo-
hydrate in relation to cold exposure. E. J.
Masoro ann C.L. Asuncion.* Dept. of Physiol-
ogy, Tufis Univ. School of Medicine, Boston,
Mass.

It was recently reported from this laboratory
that liver slices prepared from rats exposed to
0°-2°C for 1 or 2 days almost completely lost the
capacity to convert acetate-1-C™ into long chain
fatty acids. It therefore seemed important to
study the lipogenic activity of the cold-exposed
intact rat. The rats were placed in a metabolism
cage ventilated with CO2-free air; the tempera-
ture of the cage was 24°-27°C and 2°-6°C in the
case of control and cold rats, respectively. During
a 24-hr. experimental period the rat was allowed
to eat ad libitum a diet composed of 25% casein,
10% fat and 51% uniformly C-labeled glucose.
The CO. expired was collected and at the end
of the 24-hr. C4 feeding period the rat was killed
and anklyzed for fatty acid-C'* content. The
percentage of glucose-C'* converted to CO,
varied considerably but in most cases was greater
than 50% of the ingested C4. The percentage of
C* incorporated into fatty acids also showed a
considerable range. When the data are expressed
in milligrams of ingested glucose converted to
fatty acids, the cold-exposed rats showed at least
as great a lipogenic activity as did the control
animals.

409. Pressor activity of plasma on prolonged
incubation. G. M. C. Masson anp A. C.,
Corcoran. Research Div., Cleveland Clinic
Fndn., Cleveland, Ohio.

Arneil and Dekansky (1954) described a pressor
activity of incubated plasmas from children with
acute glomerulonephritis. They speculated that

FEDERATION PROCEEDINGS

Volume 1§

it might be attributable to the action of renin,
although Arneil (1955) indicated to us that it wag
not angiotonin. In our study of this phenomenon,
venous blood was sampled from patients with
essential and renal hypertension, from normo-
tensive patients and normal human subjects,
and also from normal rats, rabbits and dogg,
The plasma was immediately separated and por-
tions taken which were respectively frozen and
incubated at 37°C for 24 hr. under sterile condi-
tions. Pressor activities were tested in pithed
rats, using angiotonin as the reference standard,
The assays indicate that all plasmas yield pressor
activity on incubation. Slight pressor activity
was detected in non-incubated plasmas of two
patients with proven renal hypertension. In
general, the activity at 24 hr. of incubation corre-
sponds to about 0.08 Goldblatt pu of angiotonin,
The yield increases progressively for about 24 hr,
and does not decrease with further prolonged
(96 hr.) incubation. In contrast, added angio-
tonin disappears from incubated plasma in legs
than 24 hr. The pressor material is active in
pithed and normal rats, in dogs with sectioned
sino-aortic nerves pre-treated with tetraethyl-
ammonium and, in vitro, on the rat uterus, rabbit
duodenum and guinea-pig ileum. It is distinct
from angiotonin, vasopressin, serotonin, epi-
nephrine, bradykinin and vasotonin. The data
indicate the presence in plasma of a system,
presumably enzymatic, which forms a relatively
stable pressor agent.

410. Ketosteroid excretion patterns in the
adrenogenital syndrome. Minoru Masvupa
(introduced by Homer WuHEELON). Dept. of
Physiology and Biophysics, Univ. of Washington
School of Medicine, Seattle.

Urines from 3 adult females with congenital
adrenal hyperplasia and 2 boys diagnosed as
macrogenitosomia praecox have been analyzed
by means of a 3-step fractional hydrolysis, alumina
column chromatographic separation, and infra-
red spectroscopic identification. Quantitation
was spectrophotometric using the Zimmerman
color reaction. The ketosteroid spectra were
compared with normals of the same sex and age
range. 11-hydroxyetiocholanolone, present in
normal urines, was absent from all pathological
urines. Dehydroisoandrosterone was found in
only 2 of the 5 cases and, when found, was present
in increased amounts. 17-hydroxypregnanolone
as the p-homosteroid was found in the urines of
the 3 females, but not in the boys’ urines. The
increased titer of total ketosteroids is reflected
in increased quantities of pregnanolone, andros-
terone, etiocholanolone, 11-ketoetiocholanolone
and 11-hydroxyandrosterone. The latter showed a

lume 1§

f renin,
t it wag
menon,
ts with
normo-
ibjects,
1 dogs,
nd por-
en and
» condi-
pithed
andard,
pressor
ictivity
of two
on. In
n corre-
otonin,
t 24 hr,
olonged
angio-
in legs
tive in
ctioned
aethyl-
, rabbit
listinet
n, epi-
ie data
system,
atively

in the
[MASUDA
ept. of
hington

genital
sed as
ralyzed
Jumina
| infra-
‘itation
nerman
1 were
nd age
ant in
logical
ind in
present
inolone
‘ines of
1g. The
flected
undros-
nolone
owed a

March 1956

consistently and disproportionately high excre-
tion titer. *

41. Anemia in relation to survival following
thermal injury in the rat. Mites D. Mc-
CarTHY, MARGARET AMREIN* AND PEGGY
Coss.* Dept. of Zoology, Pomona College,
Claremont, Calif.

Male Wistar rats received back burns of 50%,
32% and 20% of total body surface by immersion
in water at 90°C for 35 sec., and within 5 min.
postburn (p.b.) were started on a minimal infusion
procedure which facilitates survival (4% body wt.
sodium lactate § mM, 14% NaCl-1.4% soln.). Simul-
taneously three groups of unburned controls were
subjected to different but limited procedures of
the total experimental regimen employed in
the burned group. Erythrocyte numbers, hema-
tocrit, and reticulocyte populations were deter-
mined. All burned animals showed decreases in
hematocrit and RBC numbers during the first
48 hr. p.b., paralleled by increased reticulocytes.
Changes in the surviving 20% burned animals
were less severe than those in the surviving 32%
and 50% burned animals. The changes in the
surviving 20% burned animals were similar to
those in the unburned but hemorrhaged controls.
However, return to preburn values was delayed
in the 20% burned animals. In all surviving burned
animals the 24-week p.b. values were depressed
compared to similar control values. No correla-
tion was found between degree of anemia and
survival. Since the number of reticulocytes seen
in all groups varied inversely as the total number
of erythrocytes, it appears that the erythropenia
following burns is not due to an inhibition of
erythrocyte formation per se, but rather to some
factor which destroys the peripherally circulating
ted cells. (Supported by the Research and De-
velopment Div., Office of the Surgeon General,
Dept. of the Army, Contract DA-49-007-MD-571.)

412. Use of D and O'8 to measure total CO;
output and body water of obese-hyper-
glycemic mice. RutH McC.uintock* AND
NatHan Lirson. Physiology Dept., Univ. of
Minnesota, Minneapolis.

The D.O'8 method for measuring total CO:
output by difference between the fractional turn-
over rates of the 2 isotopes, determined on initial
and final blood samples (Lirson et al., J. Appl.
Physiol. 7: 704, 1955) has been used in the study
of the COz production of hereditary obese mice.
This provides an index of the total energy out-
put. In 7 mice, the average discrepancy between
CO. outputs calculated by the isotope method and
those measured by collection of CO2 in NaOH was
8% (maximum, 12%). The COz outputs of 8 adult
obese mice averaged 22% higher than those of

AMERICAN PHYSIOLOGICAL SOCIETY

127

litter mate controls, under normal cage condi-
tions. The CO, outpuis of 6 young obese mice
were similar to those of controls at 6 and 9 wk.
of age, but averaged 20% more than the controls
at 12 wk. Food consumption and weight gains of
the 2 groups were measured during the CO, output
study. In the normal animals, a larger fraction
of the caloric intake is used in energy metab-
olism. In using the formula for CO output, body
water was determined by the volume of dilution
of the isotopes, or by desiccation or both. In both
obese and control mice the ratios of isotope ‘space’
to desiccation ‘space’ are more than one, and these
ratios are higher in obese mice. (Aided by a
contract between the Office of Naval Research,
Department of the Navy, and the University of
Minnesota, NR115-366.)

413. Effect of vasopressin on plasma 17-hy-
droxycorticosteroid levels. Roger K. Mc-
DonaLp AND Virerinia K. Werse.* Nail. Inst.
of Mental Health, Bethesda, Md.

The effect of a saline infusion containing com-
mercial Pitressin on plasma 17-hydroxycortico-
steroid levels was determined in a group of normal
male and female subjects between the ages of 18
and 30 years. The amount of Pitressin infused
varied between 8.7 and 31.5 units over a period of
2 hr. with the exception of two instances in which
each infusion lasted 1 hr. Control values were
obtained using saline infusions in volumes com-
parable to those of the Pitressin infusions. Blood
samples were drawn just before, during and after
each infusion. Plasma 17-hydroxycorticosteroid
levels were significantly elevated during the period
of 2 hr. in which the Pitressin was infused. A sig-
nificant fall in the 4-hr. blood eosinophile count
occurred in the subjects receiving Pitressin.
Similar studies are being conducted using highly
purified arginine-vasopressin. Elevation of the
plasma 17-hydroxycorticosteroid levels is asso-
ciated with the infusion of this preparation of
vasopressin, as well as with commercial Pitressin.

414. Comparison of intraesophageal and in-
trapleural pressures in subjects seated
and supine. J. Mreap anp E. A. GAENSLER.*
Dept. of Physiology, Harvard School of Public
Health, and 2nd and 4th Med. Service (Harvard),
Boston City Hosp., Boston, Mass.

To assess the reliability of intraesophageal
sampling of intrapleural pressure, both pressures
were recorded simultaneously in six patients.
Intraesophageal pressure was obtained from a
15 cm long latex balloon containing 1 ml of air.
Intrapleural pressure was recorded by means of
catheters introduced into the intrapleural space,
usually anteriorly in the second interspace.
Pneumothorax volumes varied from 25 to 350 ml.

128

Pressures were recorded with inductance manom-
eters. With the pressure amplitude differences
expressed as per cent deviation of intraesophageal
pressure from intrapleural pressure, the range in
six subjects seated was +14 to —18% during
quiet breathing (mean deviation —1%), +20 to
—18% during slow, deep breathing (mean devia-
tion +3%) and +7 to —5% during rapid, shallow
breathing (mean deviation < +1%). In the four
subjects studied in the supine position, the corre-
sponding deviations were +60 to +10%, +50 to
—25% and +25 to —5%, while the mean deviations
were +33, —12 and +9% respectively. The greater
deviations in the supine position relate in part
to the larger intraesophageal pressure fluctua-
tions synchronous with the cardiac cycle. In the
sitting position the mean end-expiratory pressures
relative to atmospheric pressure were: intra-
pleural pressure —5.1 cm H:0 and intraesophageal
pressure —5.7 cm HO. In the supine position
the mean end-expiratory intraesophageal pressure
was —1.1 cm H,O. The postural difference in the
pressures probably relates to compression of the
esophagus by mediastinal structures in the supine
posture.

415. Androgen and the inositol content of
male accessory organs of the rat. R. M.
Metampy AND R. B. Mason.* Agricultural
Exper. Station, Iowa State College, Ames.
The inositol assay procedure using Neurospora

crassa described by Tatum et al. (J. Biol. Chem.

163: 675, 1946) was used to study the distribution

of inositol in the male accessory organs of intact,

castrate and hormone-treated castrate rats.

Tissues were treated with 4n sulphuric acid and

autoclaved 2 hr. at 15 pounds pressure prior to

assay. Inositol expressed as ywg/gland on secre-
tion-free basis for intact males was as follows

(average of 5 animals): seminal vesicles 520;

coagulating glands 226; dorsal prostate 790; ven-

tral prqastate 327; and Cowper’s glands 40. In the
20-day castrate (tissue blotted free of fluid) these
values were 145, 41, 90, 61 and 0 respectively.

Inositol expressed as ng/100 mg tissue was highest

in the dorsal prostate (731) and lowest in Cow-

per’s (138) in the intact animal. In the 20-day
castrate the values yug/gland were as follows:
seminal vesicles 161; coagulating glands 55; dorsal
prostate 149; ventral prostate 66 and Cowper’s
glands 7. In castrates receiving 500 ug testosterone
propionate in oil daily for 20 days, the values per

gland and secretion were as follows: 11,613; 1,539;

3,471; 1,513; and 52. Seminal vesicle secretion

from intact males contained approximately

7000 wg/gm. Preliminary studies indicate that a

considerable portion of the inositol is available

to Neurospora without prior acid hydrolysis.

Seminal vesicle tissue from castrates receiving

FEDERATION PROCEEDINGS

Volume 1§

androgen for 60 hr. was higher in inositol (27%)
in fasting animals fed this substance by stomach
tube than in fasting controls. (Supported by
Office of Naval Research, NR 164-144.)

416. Three phase counter-current distribu-
tion. HerBert L. MELTZER (introduced by
Earu T. ENGLE). Depts. of Biochemistry, New
York State Psychiatric Inst., and College of
Physicians and Surgeons, Columbia Univ., New
York City.

A technique has been devised which permits
solute to be distributed among 3 liquid phases in
a pattern analogous to that described by Craig for
two phase counter-current distribution. Top and
middle phases are each caused to move counter-
current with respect to bottom phase and at right
angles with respect to each other, within a pattern
bounded by an equilateral triangle. An automatic
apparatus has been constructed to carry out a
10-transfer distribution according to this pattern,
Solute is placed in the apex tube, solvent is placed
in reservoirs along each side, and multiple samples
are withdrawn at the base. The mathematics of
this system and the preliminary results obtained
with brain lipides wil. be discussed. (Supported
in part by research grants from the Natl. Inst. of
Neurological Diseases and Blindness, PHS, and
Multiple Sclerosis Soc.)

417. Direct effect of ACTH on capillary perme-
ability in inflammation. VAaty MENKIN.
Agnes Barr Chase Fndn. for Cancer Research,
Temple Univ. School of Medicine, Philadelphia,
Pa.

Earlier studies have indicated the presence of

2 factors in inflammatory exudates concerned in

the mechanism of increased capillary permeability

with inflammation: 1) leukotaxine primarily in
the initial stage of the reaction, 2) exudin in the
later or acid stage. Leukotaxine is repressed by
cortisone and hydrocortisone. Exudin is inhibited
by ACTH. The rapid repressive effect on capillary
permeability by ACTH in cutaneous areas treated
with an acid exudate or its contained exudin have
suggested that the effect may be a direct one
without the mediation of the adrenal cortex. The
extensive local increase in capillary permeability

in inflammation lends support to the view that a

certain amount of ACTH injected into the circu-

lation may seep directly into the inflamed area
and, there, exert its suppressing effect on exudin.

Further evidence indicates that repeated daily

injections of ACTH directly into an acutely in-

flamed area gradually represses the effectiveness
of either the acid exudate or of the recovered
exudin in increasing capillary permeability.

Adrenalectomized rats display a similar repres-

sive effect on capillary permeability when exudin

—— oo

—_—or as Sw <a

—m™ ere pmo ss ah Sf

‘ume 16

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»x. The
ability
that a
> circu-
d area
exudin.
1 daily
ely in-
iveness
-overed
ability.
repres-
exudin

March 1956

is injected, into the dermis together with ACTH.
Accessory adrenal structures have rarely been
encountered. Astwood’s purified fraction of corti-
cotropin has also induced an inhibitory effect on
the effectiveness of exudin. Finally, and most
important, Li’s a-corticotropin, a single chemical
entity, likewise suppresses the increased capillary
permeability induced by an acid exudate or exu-
din. The aggregated observations therefore
indicate that ACTH acts directly in suppressing
the increased capillary permeability induced by
exudin, liberated in turn by injured cells at the
site of inflammation.

418. Cardiac glycogen fractions following
ligation of coronary artery of the dog.
Artuur W. Merrickx* anp Datuas K. MEYER.
Dept. of Physiology and Pharmacology, Univ. of
Missouri School of Medicine, Columbia.

The anterior descending branch of the left
coronary artery of 12 adult dogs was ligated
approximately 2 cm below the point of origin.
Four hours later the heart was removed and the
left ventricle sectioned. A strip of tissue was
removed in a lateral-superior direction starting
1 cm below the ligature and on the coronary
artery. This strip was divided into approximately
6 equal parts which were labeled zones A (anox-
emic area) through F (normal area). The free
and bound glycogen fractions were determined
from each zone by conventional methods. Pre-
vious work (Sa¥YEn et al. J. Clin. Investigation,
30: 932, 1951 and Poppen et al. J. Histochem. and
Cytochem. 1: 160, 1953) has suggested 3 zones may
be observed polarographically and histochemically
following coronary ligation: infarcted, border
and normal. We have confirmed this by chemical
means. Our results show there is the expected
decrease in total glycogen (TENNANT et al. Am.
Heart J. 12: 168, 1936) in the zone of infarction
but the ratio of bound to free glycogen was un-
changed from the normal (71.8% free glycogen
in the infarcted area compared to 68.8% in the
normal zone). However, the border zone showed
a preferential depletion of the free glycogen
(50.9%). The implication is that in an ischemic
area free glycogen is utilized more readily than
the bound glycogen, or the bound glycogen is
Maintained at the expense of the free glycogen.
(Supported in part by a grant from the Dazian
Foundation.)

419. Presence of homologous cells in the
marrow of mice protected from the effects
of irradiation by homologous marrow.
Rutu M. Merwin anp CHARLES C. ConGpon
(introduced by Wiuure Smits). Natl. Cancer
Inst., Natl. Insts. of Health, Bethesda, Md., and

AMERICAN PHYSIOLOGICAL SOCIETY

129

Biology Div., Oak Ridge Natl. Lab., Oak Ridge,

Tenn.

It is not known whether bone marrow injected
intravenously into mice that have received whole-
body irradiation prevents the death of these mice
by repopulating the marrow or by some other
mechanism. Using the following method, the
presence of the injected cells or their progeny in
the marrow of the protected mice could be deter-
mined and some quantitative measure made.
Homologous marrow was used to protect irra-
diated LAF mice and at various intervals there-
after marrows of the protected mice were tested.
The marrows were tested by injecting them subcu-
taneously into LAF mice each of which carried
a nonvascularized graft of tissue from the homol-
ogous strain of mice. The presence of homologous
cells was indicated by the disintegration of the
homograft (J. Nat. Cancer Inst. 14: 819-839). It
was found that marrows tested at 6 hr.—4 days
after protection caused 70% of the grafts to
disintegrate whereas marrows tested at 12 days,
at which time the marrows of irradiated and
protected mice have usually completely regen-
erated, caused 100% of the grafts to degenerate
and, further, degeneration occurred sooner.
Marrows tested at 60-180 days caused a response
similar to those tested at 12 days. The proportion
of the marrows of the protected mice that is
occupied by the homologous cells is being studied.
Spleen, blood, lymph nodes and thymus of mice
that had been protected for 60-180 days also
contained homologous cells.

420. Bleeding time studies after dextran
infusions in normal and irradiated dogs.
Sot M. Micuaetson,* ANDREW KoNNERTH*
AND JoE W. How.tanp. Dept. of Radiation
Biology, School of Medicine and Dentistry, Univ.
of Rochester, Rochester, N. Y.

Recent clinical observations on the use of
dextran as a plasma substitute in the dog reveal
a similar tendency toward prolonged bleeding
previously observed in man. Bleeding time and
platelet level measured 4-5 hr. after a single
infusion of a clinical dose of dextran in the dog
were within normal limits. After a third or fourth
weekly infusion a slight prolongation of bleeding
time occurred without alteration in platelet con-
centration. In dogs receiving whole body x-radia-
tion (Lp-50/30) a marked prolongation in bleeding
time started 1 wk. post irradiation. Among such
dogs receiving single infusions of clinical doses of
dextran, no evidence of an enhancement of the
bleeding time prolongation was observed. Daily
infusions of clinical doses of dextran resulted in
prolonged bleeding times following the third
infusion. Increasing the dose or concentration of

130 FEDERATION PROCEEDINGS

dextran indicated that this bleeding tendency is
dose and concentration dependent. The rate of
infusion showed little influence on the observed
bleeding tendency. Reports indicate that bleeding
time is dependent on the integrity of both vessel
wall and platelets. In the reported experiments
there is no evidence of a correlation between
bleeding time prolongation and a decrease in
platelet numbers following dextran infusion.
Clotting time and clot retraction were unchanged.
Erythrocyte sedimentation rate was increased in
each case. It is suggested that the bleeding tend-
ency, when it does appear, is induced by an im-
pairment of the vascular integrity. Epinephrine
and Pitressin counteract the tendency toward
prolonged bleeding which follows dextran
infusion.

421. Effect of K vitamins on prothrombin
and proconvertin levels of cholecystneph-
rostomized dogs. G. J. Miuuar,* L. FisHer,*
AND L. B. Jaques. Dept. of Physiology, Univ.
of Saskatchewan, Saskatoon, Canada.

Chronic bile lack in cholecystnephrostomized
dogs: leads to the development of a bleeding
tendency. Insufficient absorption of the fat solu-
ble vitamins K is presumed to occur which in
turn causes a depression of the coagulation factors
prothrombin and proconvertin. A comparison of
the relative effectiveness of the K vitamins ad-
ministered orally and intravenously in restoring
the plasma levels of prothrombin and procon-
vertin has been studied. Vitamin K; and Syn-
kavite were found to be more effective and rapid
in returning prothrombin and _proconvertin
values to normal than vitamin K; when given
orally on an equimolar basis. Intravenously, on
the other hand, vitamins Ki, Ke and K; had
approximately the same degree of biological
activity in correcting proconvertin levels but the
therapeutic effect obtained was less prolonged in
the case of vitamin K;. An intravenous equimolar
dose of Synkavite had only a transient elevating
effect on proconvertin values when compared
with the other three preparations.

422. Factors in decline of basal metabolism
with age. A. T. MILueR, Jr. ann M. C.
Conrap.* Dept. of Physiology, Univ. of North
Carolina Med. School, Chapel Hiil.

Basal metabolism, response to propylthiouracil
and thyroxine, and body composition were de-
termined in albino rats ranging in age from 17-174
days. The decline in basal metabolism with age
was eliminated by expressing metabolism in
terms of weight 0.5. The increase in fat and bone
content with age was offset by a decrease in extra-
cellular fluid volume (chloride space) so that the
‘active tissue mass’ was essentially unchanged.
The percentage depression of BMR by propyl-
thiouracil was greater in old than in young ani-

Volume 16

mals, and animals treated with PTU daily begin-
ning at 27 days of age had the same rate of decline
in metabolism with age as did normal animals,
It may be concluded that changes in body com-
position and in thyroid function play little or no
part in the age-specific decline in basal metab-
olism. Calculations based on data in the literature
indicate that decrease in the relative weight of
the viscera may account for about 50% of the
decline in basal metabolism with age and tissue
Qo, values, recalculated to a wet weight basis,
suggest that decline in the respiratory rate of
certain tissues may account for an additional
10-15%. The factors responsible for the remainder
of the age-specific decline in metabolic rate are
as yet undetermined. (Aided by a grant (No. H-
818) from the Natl. Inst. of Health, PHS.)

423. Inhibition of PMS by various poly-
hydroxybenzene derivatives. NaTHAN MI11-
MAN AND FRED Rosen.* Ortho Research Fndn.,
Raritan, N. J.

Recent studies in these laboratories and else-
where (NOBLE AND GRAHAM. Federation Proc. 13:
106, 1954) have indicated the in vitro inactivation
of pregnant mare serum gonadotrophin (PMS) by
2,6-dimethylhydroquinone (DMHQ) and related
compounds. The present report details some of our
findings on the mechanism by which the ovarian
weight-stimulating activity of PMS is destroyed
in the presence of certain quinones and poly-
hydroxybenzene derivatives. Tris buffer, glycine,
hydroxyproline, serine and the essential amino
acids, with the exception of lysine, were found to
block the action of DMHQ on PMS, while other
amino acids and various primary, secondary and
tertiary amines were without effect. The
d-catechin and quercetin were previously reported
(Ros—EN AND MiuuMan. Endocrinology 57: 466,
1955) to be inactive against PMS. It was deter-
mined, however, that if these substances were
tested in the presence of 0.04 m Mn*, or incu-
bated with PMS at px 10, they became capable of
inhibiting the gonadotroph. Similar treatment of
DMHQ with Mn** significantly increased the rate
of in vitro inactivation of PMS. In vivo tests indi-
cated Mn-treated d-catechin did not affect the
estrous cycle of rats. Other experiments were
concerned with the inhibition of PMS by the
oxidized form of DMHQ, and with the analysis of
spectrophotometric data on the PMS-DMHQ
system.

424. Extent of the effective ‘vulnerable period’
for transthoracic, high-energy capacitor
shocks in dogs. W. R. MILNoR anv W. B.
KOUWENHOVEN (introduced by Davip Gros).
Schools of Medicine and Engineering, Johns
Hopkins Univ., Baltimore, Md.

Capacitor discharge shocks were given to

ume 1§

begin-
lecline
‘imals,
- com-
, Or no
netab-
rature
ght of
of the
tissue
basis,
ate of
itional
ainder
te are
Vo. H-
3.)

poly-
MIL1-
F'ndn.,

1 else-
oc. 13:
vation
(S) by
elated
of our
varian
troyed
poly-
ycine,
amino
ind to
other
"y and
The
ported
: 466,
deter-
. were
incu-
ble of
ent of
e rate
3 indi-
>t the
were
y the
ysis of
IMHQ

sriod’
acitor
W. B.
‘ROB).
Johns

an to

March 1956

anesthetized dogs with normal heart rhythm
through two copper electrodes, 4 in. square, one
on each side of the thorax. Inter-electrode resist-
ance varied from 500 to 900 ohms. Shocks from
capacitors of 25-50 uf., charged to 3000 to 4000 v.
produced bursts of rapid ventricular extrasystoles
in 104 of 112 trials, leading in 7 instances to ven-
tricular fibrillation. Susceptibility to this effect
was not restricted to the relatively short ‘vulner-
able period’ observed when stimuli of lower volt-
age are applied directly to the heart. Multiple
ventricular extrasystoles were produced by shocks
applied in any portion of the heart cycle. Ventricu-
lar fibrillation occurred after shocks which fol-
lowed the QRS complex by intervals ranging from
4% to 65% of the control R-R interval. Similar
effects reported by Moe et al. (Am. J. Physiol.
134: 473, 1941) using direct epicardial stimuli were
attributed to persistent myocardial depolariza-
tion. No evidence of such a phenomenon appeared
in peripheral or intracardiac ECG leads in our
experiments, but this possibility is still being
investigated. These results indicate that the
effective vulnerable period, defined as that portion
of the heart cycle during which a single stimulus
can bring about ventricular fibrillation regardless
of latency between stimulus and response, may
under some conditions in the intact animal extend
throughout systole and at least the first 4 of
diastole.

425. First physiological observations with
the human gradient calorimeter. Davip
Minarp, T. H. BENZINGER AND CHARLOTTE
KirzincER.* Naval Med. Research Inst., Be-
thesda, Md.

Human gradient calorimetry permits continuous
recording of rapid changes in heat output from
the adult human at rest or working under selected
environmental conditions. The supine subject
rests upon a light metal frame suspended within
the chamber by nylon cords. In preliminary
studies, the subject performed the following
separate Maneuvers: exercise using a spring
expander; hyperventilation; change in posture.
Heat loss was recorded continuously before, dur-
ing and after each maneuver. Conditions were
29.5°C air temperature, 30% relative humidity,
and 800 1/min. air flow. Heat output rose from 31
gm cal/sec. at rest to 55 gm cal/sec. during 220
sec. of exercise. During recovery the output
dropped rapidly from this peak for 5 min. and
then more slowly in stepwise fashion to the resting
level. Excess heat amounting to 9800 gm cal.
appeared during work and recovery, 95% being
evaporative. During 110 sec. voluntary hyper-
ventilation and recovery, 2100 excess calories
were measured. Again the major fraction was
evaporative. The recovery curve was smooth
thus differing from the curve in exercise. Reducing

e422 tha

AMERICAN PHYSIOLOGICAL SOCIETY 131

radiative surface area by changing posture from
supine extension to flexion resulted in a prompt
drop in calories measured by the main chamber.
The ventilatory circuit (evaporative output)
showed no change. During 5 min. in the flexed
posture, 2200 cal. were stored and appeared as
excess heat upon resuming the extended position.

426. Protein metabolism in Ehrlich’s mouse
ascites carcinoma cells. Kivie MoLpAve
(introduced by Marta SEeRAYDARIAN). Dept.
of Biochemistry, Tufts Univ. School of Medicine,
Boston, Mass.

Recent studies (Hoaness et al. Biochem. et
biophys. acta 16: 99, 1955) have demonstrated that
$*-labeled H. coli proteins do not significantly
contribute to the synthesis of the induced enzyme
B-galactosidase. These results have been inter-
preted as being incompatible with the classical
view proposed by Schoenheimer for the dynamic
state of proteins within the cell. The implied
irreversibility of protein synthesis has been
investigated in ascites cells. Suspensions of
ascites cells previously labeled in vivo with C™
alanine or lysine were incubated in vitro for 9
hr. During this time, the C4 content of the lipide
fraction increased slightly, that of the nucleic
acids remained unchanged and the specific ac-
tivity of the proteins decreased 10-17%. Approxi-
mately 12% of the protein-bound radioactivity
from such preparations was recovered as free
amino acids. Implantation of similarly labeled
cell suspensions in dialysis sacs into the peritoneal
cavity of normal mice resulted in a 2-fold increase
in the number of cells within 48 hr. No gain in the
dry weight of the cells occurred. Under these
conditions, the nucleic acids lost 22-38% of their
radioactivity and the specific activity of the
proteins decreased 40-50%. The decrease in the
specific activity of the protein, that is the loss of
radioactivity without concomitant loss of protein,
and the appearance of radioactive free amino
acids suggest that proteins exist in dynamic
equilibria within the cell. (Supported in part by
Grant DRG-342 from the Damon Runyon Me-
morial Fund.)

427. Passage of potassium from gastric con-
tents to blood stream. Hanns C. Mout,
CuHaRLEs F. Copr aNnp ALAN L. Orvis (intro-
duced by Epwarp J. Batpgs). Mayo Fndn. and
Mayo Clinic, Rochester, Minn.

The finding that the absorption of sodium from
the stomach is blocked when acid is present led
us to ascertain whether or not the passage of
potassium from gastric contents to blood stream
is similarly effected by hydrogen ions. Dogs
anesthetized with pentobarbital sodium were used.
The antral mucosa was always excluded by liga-
tion. Water labeled with deuterium or tritium

132

and potassium as K‘* were employed. With the
gastric mucosa at rest and no acid being secreted,
potassium as well as sodium was absorbed. The
rate of passage of K*? from gastric contents to
blood stream was 6-9% of that present in the
contents per hour, which is considerably slower
than that noted previously for sodium (12-35%/
hr.). However, unlike sodium, the rate of passage
of isotopic potassium from the gastric contents
into the blood stream was unaffected by the
presence of extrinsic (added) or intrinsic (se-
creted) hydrochloric acid. Nor was the rate
dramatically affected by the addition of potassium
hydroxide or sodium hydroxide. The experiments
suggest that the absorption of potassium and
sodium from the stomach is, in the main, inde-
pendent, and that since the passage of water from
gastric contents to blood stream was not affected
significantly by any of the changes made in the
gastric contents, its passage from gastric con-
tents to blood is likewise independent of both
sodium and potassium.

428. Body temperature during work in man
on restricted water intake and low calorie,
earbohydrate diet. J. EpGgar Monaa1z,*
Francisco GRANDE,* E.swortH BuskIRK,*
JoseEPH Brozex, Henry LONGSTREET TAYLOR
AnD AncEL Keys. Lab. of Physiological Hygiene,
Univ. of Minnesota, Minneapolis.

Rectal temperatures of 12 clinically healthy
soldiers were measured during a 1-hr. walk on a
motor-driven treadmill at 3.5 miles/hr. on a 10%
grade before (control), during (experimental) and
after (recovery) a period of water and calorie
restriction. The conditions were rigidly controlled
and the men lived and worked in an environment
of 78°F and 65% relative humidity. The daily
water intake during the experimental period
was 900 cc for each of 6 men (group J) and 1800
ec for the other 6 (group IZ). Each man in both
groups @eceived 1000 cal./day of pure carbo-
hydrate as the only food and used 1200 cal. for
2 hr. of treadmill work daily. In group I, there was
a continuous increase in rectal temperature at the
end of 1 hr. work until, after 5 days of water
restriction, the average was 1.6°C higher than
before water restriction. In group II only a small
increase of 0.6°C over the value before water
restriction was found on the 3rd day and by the
6th day this value had returned to prestarvation
levels and remained essentially unchanged to the
end of the water restriction period. Administra-
tion of water ad libitum to group I brought tem-
peratures back to the prerestriction levels and
produced no important change in group IT. It is
concluded that the water deficit in group IT was
insufficient to produce a persistent alteration in
thermoregulation as observed in group I.

FEDERATION PROCEEDINGS

Volume t§

429. Extractable heparin level in different
animal tissues. FRANK C. MonxuouseE. Dept,
of Physiology, Univ. of Toronto, Toronto, Canada,
One of the difficulties in solving problems

relevant to the physiology of heparin is the lack of

methods suitable for use with small quantities of
tissue. The following procedure gives reasonably
consistent results with 2-100 gm of tissue. The
tissue is macerated in 5 ml of 0.5 m KSCN/gm of
tissue, and the mixture agitated for 3 hr. The
extract is filtered through gauze and treated
with 4 volume of 80% phenol, and allowed to
stand overnight. After centrifugation, the heparin
is precipitated from the aqueous layer by adding3
volumes of ethanol and allowing it to stand for

2 hr. at 4°C. The centrifuged precipitate is suc-

cessively washed with ethanol and ether and

dried. The heparin is extracted from the precipitate
with saline and assayed. Further purification
can be secured by reprecipitating with alcohol,

dissolving in saline and precipitating with 5%

benzidine HCl in water. Heparin levels have been

determined in a variety of tissues from rabbit, dog,
beef, monkey and man. The levels expressed in

units/100 gm of tissue varied from less than 40

in the rabbit intestine to 4800 u in the liver of a

hypophysectomized dog. Radioactive heparin

was obtained from dog liver 48 hr. after the
animal had been fed S* contained in hydrolyzed
yeast.

430. Changes in lung compliance during
development of ANTU pulmonary edema.
JaMEs C. Moore anp Marx 8. SExTER (intro-
duced by Hamppen C. Lawson). Dept. of
Physiology, Univ. of Louisville School of Medi-
cine, Louisville, Ky.

Lung compliance was measured in 15 anesthe-
tized dogs at frequent intervals following the slow
intravenous injection of 40 mg/kg of ANTU (2%
in propylene glycol). Clinically recognizable
pulmonary edema developed in 12 animals. Death
occurred in 20 min-6 hr. Animals developing
pulmonary edema showed consistent decreases in
lung compliance, beginning usually less than 1 hr.
after the ANTU injection. Compliance decreased
slowly at first, more rapidly in the terminal stages.
At death, the compliance had been reduced to
about 3-3 of the control value. No sustained or
temporary decreases were found in those animals
in which pulmonary edema failed to develop.
Three to 6 cardiac outputs (T-1824) were done in
each of 7 of these dogs. Calculated ‘central blood
volume’ decreased progressively throughout each
experiment in all dogs receiving ANTU, including
2 which did not develop pulmonary edema. In
an additional series of experiments, animals were
killed at various compliance levels and portions
of the lungs were examined microscopically.

ee a Ae ee ee ee ee ee ee ee ee Sh Ne eo ee se a

olume 16

ifferent
E. Dept,
Canada,
roblems
2 lack of
tities of
Sonably
ue. The
N/gm of
hr. The
treated
wed to
heparin
vdding 3
and for
is suc-
ler and
cipitate
fication
alcohol,
ith 5%
ve been
it, dog,
ssed in
lan 40
rer of @
heparin
ter the
rolyzed

during
-dema.

(intro-
ept. of
, Medi-

nesthe-

March 1956

Perivascular edema was found in animals killed
early, with moderate changes in compliance.
Animals killed in later stages, after a more marked
decrease in compliance had been observed, showed
greater amounts of perivascular edema and some
interstitial edema. Only animals killed in the
terminal stages, with alveolar edema as well,
showed clinically recognizable signs of pulmonary
edema. (Supported in part by PHS Grant H-1585.)

431. Michaelis-Menten kinetics re-visited.
ManvE. F. Moraes. U. S. Naval Med. Re-
search Inst., Bethesda, Md.

While the simple mathematical analysis corre-
sponding to the Michaelis theory of enzyme action
is widely known and used, certain of its implica-
tions and consequences have received little atten-
tion. The author will summarize some recent
theoretical investigations (by several others as
well as by himself) concerning, 1) conditions
under which steady-state measurements alone
virtually assume that the ‘Michaelis Constant’ is
an equilibrium constant; 2) behavior of an enzyme-
substrate system in the transient (non steady-
state) state; and 8) assumptions involved in
considering the 2nd step of the Michaelis-Menten
reaction scheme to be ‘irreversible.’

432. Support of the circulation with Aramine
during high levels of positive pressure
breathing in the dog. W. L. Morgan, J. T.
Binron, S. J. Sarnorr (introduced by DaNnriBL
A. McGinty). Lab. of Cardiovascular Hemo-
dynamics, Natl. Insts. of Health, Bethesda, Md.
Seventeen anesthetized dogs were subjected to

high levels of intermittent positive pressure

breathing (PPB), which resulted in marked hypo-

tension. Seven dogs partially protected by a

counter-pressure suit had an average airway

pressure of 69 cm of water, and 10 dogs without
counterpressure where subjected to an average
airway pressure of 43 cm of water. With airway
pressure held constant, the femoral artery pres-
sure progressively declined. When, after the onset
of PPB, the effective arterial pressure (femoral
artery pressure minus right atrial pressure) had
dropped to low levels (to 11-46 mm Hg) within
2-22 min., Aramine was given in doses at 0.2
mg/kg i.v. In 8 dogs, it was given intramuscularly
in doses of 0.5 mg/kg. In every instance the blood
pressure rose to substantially higher levels en-
abling the animals to sustain the PPB for an
average of 70 min. compared to 10 min. before
the drug was given. An average of 3 injections of

Aramine was given per animal. Aramine provided

significant circulatory support whether or not

counter-pressure was used but was of somewhat
shorter duration with counter-pressure perhaps
because of the higher levels of PPB used. It is

AMERICAN PHYSIOLOGICAL SOCIETY

133

proposed that this vasopressor drug provides
support to the circulation by a) constricting
peripheral vessels, including veins, and thereby
replacing displaced blood into the lung and
b) elevating the ventricular function curves; this
influence thereby counteracts the tamponad
effect of high levels of PPB.

433. Activation of cerebellar cortex by afferent
impulses. F. Morin. Wayne Univ. College of
Medicine, Detroit, Mich.

When recorded monopolarly from the surface of
anterior and paramedian lobule the potential
evoked by threshold stimulation of superficial
radial nerve usually consists of a fast, low ampli-
tude, positive deflection (potential z) followed
by a diphasic (plus-minus) wave (potential y).
In non-anesthetized preparations increasing the
stimulus strength usually results in the appearance
of other waves which follow potential y. These
later waves are sometimes observed also in
nembutalized cats. Potential x follows high rates
of peripheral stimulation, is more resistant than
potential y to anoxia, anesthesia, local cooling,
local applications of anesthetics. Its amplitude
increases when a needle electrode reaches the
granular layer, it decreases or disappears when
bipolar surface electrodes are employed and the
interelectrode distance is reduced. Potential x
does not seem to be related to any specific spinal
tract or more to deep rather than cutaneous
receptors. Potential y is similar to the evoked
potentials of sensory cortex. The positive phase
is more resistant than the negative phase to
topical cooling. Topical application of strychnine,
procaine, Nembutal, acetylcholine, and veratrine
affects the two phases of potential y in a manner
similar to that known for the cerebral evoked
potentials. Potential y appears to be due to
activity of the Purkinjie cells following the ar-
rival of impulses in the granular layer indicated
by potential x. The observations so far available
suggest that in the production of y potentials
Purkinjie cell dendrites play a major role. (Sup-
ported by PHS Grant, No. B405(C).)

434. Comparison of polarographic results with
standard tests of tryptic digestion of
plasma proteins. Otro H. Mi.uer anp
Irsuro Yamanoucni.* Dept. of Physiology,
State Univ. of New York College of Medicine,
Syracuse.

Human and bovine albumin and y-globulin
fractions were digested for 5 hr. at 37°C in a
pH 7.1 phosphate buffer with several commercial
trypsin preparations. At appropriate time inter-
vals samples were removed for the following
analyses: a) formol titration, b) polarographic
analysis in a divalent cobalt buffer, c) measure-

134

ment of optical density of a trichloracetic acid
filtrate at 280 my and d) polarographic analysis of
a sulfosalicylic acid filtrate in a trivalent cobalt
buffer. From graphs in which these data were
plotted against time, the following conclusions
could be drawn: 1) Each analysis measures some-
thing different from the other. 2) Corresponding
human and bovine proteins behave essentially the
same. 3) Commercial trypsin preparations of
comparable activity differ from each other in ways
that can be characterized polarographically. 4)
Trypsin digests albumin without reaching a
limit in 5 hr.; values obtained in analyses a)
and b) increase in accordance with Schiitz’s law.
Split products revealed by analysis c) increase
similarly, but those revealed by analysis d) are
produced only within the first hour of digestion.
§) Trypsin has hardly any effect on freshly dis-
solved y-globulin as indicated by analyses a) and
b). During 5 hr., there are no split products
demonstrable by analysis d) and only a small
number after 2 hr. demonstrable by analysis c).
Aging in the refrigerator renders the solution of
y-globulin much more susceptible to the action
of trypsin. (Supported by a grant from the Na-
tional Insts. of Health, PHS.)

435. Effects of induced cold torpor on hemo-
concentration in Chrysemys picta. X. J.
Musaccuta AND M. L. Srevers.* Dept. of
Biology, St. Louis Univ., St. Louis, Mo.
Experiments were designed to determine the

effects of induced cold torpor on the blood con-

centration of the turtle, Chrysemys picta. In one
experiment, 20 specimens were used as controls
and 63 were exposed to 4°+4°C. At intervals of

1, 2 and 3 months, 20 specimens were killed,

and the following analysis made: hematocrit,

whole blood specific gravity, plasma specific
gravity and plasma protein (by calculation.) The
hemotocrits of the controls were 31.9+0.67 and
those from the turtles in cold torpor were 26.0+-

1.50, 24.041.95 and 20.0+1.47 for the 1, 2 and 3

months respectively. The whole blood specific

gravity was significantly altered from 1.0423+

0.0009 in the controls, to 1.0380+0.0017 and

1.0344+0.0013 in specimens after 2 and 3 months

in cold torpor. There were no significant changes
in the plasma specific gravity and plasma protein.

It was concluded that long-term cold torpor

resulted in hemodilution. A series of short-term

experiments with turtles in rapid induced cold
torpor were also carried out. Twenty specimens
whose body temperatures were maintained at 0°C
for 1 wk. showed a 20% decrease in hemotocrits.

Twenty specimens whose body temperatures were

rapidly lowered to 0°C and then returned to room

temperatures of from 19°-22°C also showed
hemodilution. In all of the short-term experi-

FEDERATION PROCEEDINGS

Volume §

ments, there was no significant change in plasma
specific gravity or plasma protein concentrations,
The experiments were also designed to evaluate
the effects of repeated bleedings on turtles in both
the control and experimental groups. It was found
that repeated bleedings (1-1.5 cc at 3-hr. intervals)
resulted in hemodilution in both control and
experimental animals.

436. Contralateral mnemonic effects with
ipsilateral sensory inflow. R. E. Myers*
AND R. W. Sperry. Div. of Biology, California
Inst. of Technology, Pasadena.

Simple visual discriminations learned with one
eye are retained with the other eye by cats having
all crossed optic fibers destroyed at the chiasma,
To find out whether or not the mnemonic traces
involved are confined entirely to the trained
(ipsilateral) hemisphere, 14 chiasma-sectioned
cats were taught 1 or 2 visual pattern discrimina-
tions with a mask covering one eye. On completion
of training, varying portions of cortex were
removed from the hemisphere on the trained side.
These removals varied from restricted ablation
of the visual areas I plus IJ, to complete cortical
ablation extending forward to the edge of the
posterior sigmoid and coronal gyri. Following a
postoperative rest period of 11-24 days, the cats
were tested for retention with the untrained eye.
The results cbtained appeared not to be affected
by the extent of cortical removal beyond the
minimal lesion, but did differ for the 2 discrimina-
tions used. In the case of the less difficult dis-
crimination (horizontal vs. vertical striations)
there was nearly perfect retention in 5 instances,
partial loss in 7, and apparently complete loss in
only one. In both of 2 cases where the more difficult
discrimination (solid circle vs. open ring) was
used, transfer to the untrained eye was com-
pletely absent, although this same discrimination
has been shown to transfer at a high level in the
absence of cortical lesions. The findings demon-
strate a mnemonic carryover via the corpus
callosum into the hemisphere not directly re-
ceiving the sensory information. The carryover
was sufficient to effect partial to complete re-
tention of simple discriminations but was not
sufficient to support the performance of more
difficult discriminations.

437. Influence of acute hypoxia on pulmonary
circulation of dogs during apnea. G. G.
Nanas (introduced by GrorcE Faur). Dept. of
Physiology, Univ. of Minnesota, Minneapolis.
The purpose of this study was to investigate the

influence of moderate hypoxia on the pulmonary

circulation after eliminating the ventilatory
response to hypoxia and its secondary effects on

the circulation. After pentothal anesthesia, 10

age OR Gia oe a ee ee” eee a, ee ea a a Ye ee

lume 15

| plasma
trations,
evaluate
3 in both
as found
tervals)
rol and

S with
Myers*
ilifornia

vith one
3 having
hiasma,
c traces
trained
»ctioned
‘rimina-
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xX were
ed side,
rblation
cortical
of the
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he cats
ed eye,
affected
nd the
rimina-
ult dis-
iations)
stances,
loss in
difficult
g) was
s com-
ination
| in the
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tly re-
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ete re-
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1onary
G. G.
Dept. of
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ate the
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March 1956

dogs were ‘fitted with catheters in the pulmonary
artery and vein, right atrium and descending
aorta. Respiratory arrest was induced by curare-
like compounds and mechanical breathing in-
stalled. Photokymographic records of pressures
and cardiac output determinations were made
during artificial respiration and at the end of
90-sec. periods of ‘apneic oxygenation’ and
‘sypneic hypoxia.’ ‘Apneic oxygenation,’ is a
period of respiratory arrest following denitrogena-
tion of the animal and during which the trachea is
connected to a reservoir containing 100% Os.
‘Apneic hypoxia’ is a period of respiratory arrest
following ventilation with room air. After 90 sec.
of ‘apneic oxygenation’ mean pulmonary artery
and vein pressure, and pressure gradient between
pulmonary artery and vein were significantly
lowered. Mean femoral artery was significantly
increased. Cardiac output and calculated pul-
monary and peripheral resistance were not
changed. During ‘apneic hypoxia’ (with arterial
oxygen saturation in the vicinity of 50%) mean
pulmonary artery, pulmonary vein and femoral
artery pressure and pressure gradient between
pulmonary artery and vein were significantly in-
creased. Calculated pulmonary and _ peripheral
resistance rose significantly from 2.5-3.6 and
45-54 mm Hg 1/min. respectively. These increases
occurred in the presence of a significant fall in
heart rate and in right atrial pressure, and while
cardiac output did not change significantly.

438. d-Glucosamine metabolism by rat tis-
sues in vitro. Henry I. NAKADA AND JACK
B. WoLFE (introduced by Dovetas R. Drury).
Scripps Clinic and Research Fndn., La Jolla,
Calif.

Using the early work of Lutwak-Mann on the
enzymic decomposition of aminosugars as a
starting point, the catabolism of d-glucosamine by
tat tissues has been studied. Using radiocarbon-
labeled glucosamine, rat tissue slices oxidized
this aminosugar to CO, in the following relative
order: brain (100), kidney (88.5), testis (79),
lung (74), spleen (68.5), heart (16.3), muscle
(diaphragm) (10.7) and liver (5.7). Glucosamine
had little or no effect on glucose-U-C"* oxidation
by brain or kidney slices; but, in the presence of
glucose, these tissues could produce little or no
NH; or radioactive CO, from glucosamine. How-
ever, when rat brain slices were incubated with
glucosamine-6-phosphate, added glucose had no
appreciable effect on ammonia production from
this phosphorylated aminosugar. These results
point out that hexokinase is a common step in the
metabolism of both glucose and glucosamine and
clearly demonstrates the necessity of phosphory-
lation as a step prior to deamination. Rat brain
slices can also deaminate glucosamine-6-phos-

AMERICAN PHYSIOLOGICAL SOCIETY

135

phate under anerobic conditions, showing that the
deamination of glucosamine-6-phosphate is not
an oxidative reaction. A partial purification of the
enzyme system responsible for the deamination of
glucosamine is being attempted.

439. Stress response in a_ poikilotherm.
Rotanp M. NarponE AND RicHarpD St. JOHN
(introduced by C. Tum SupEn). Dept. of Biology,
Catholic Univ., Washington, D.C.

A study was made to ascertain whether the
Selye stress syndrome, as indicated by an eosino-
penia, occurs in a poikilotherm. The experimental
organism used was the turtle Pseudemys elegans
and the eosinophil level of the blood after epineph-
rine injection, cold exposure and cortisone injec-
tion was used as the indicator. Because too fre-
quent removal of blood from a turtle created a
stress all experiments were standaridzed with
counts made immediately before application of a
stress and 4} hr. later. Epinephrine injection
produces an eosinophilia, possibly based on the
sympathetic effect of this compound on bone
marrow. The average increase was 37.4%, +10.2%.
A low temperature environment produces an
eosinopenia. This indicates the presence of the
hormonal mechanism of the stress syndrome.
The average fall was —37.5%, +3.1%. A com-
bined treatment of epinephrine injection and cold
stress produces an eosinopenia. This is possibly
brought about by the suppression of the eosino-
philic effect of epinephrine. Treatment with
cortisone produced an eosinophilia. The results of
this experiment were compared to the experiment
with epinephrine and to other work. Based on a
comparison with homeotherms in a cold environ-
ment, the adrenal-pituitary mechanism of the
Selye stress syndrome is active in the turtle in
this same environment.

440. Reduction in serum complement con-
centration in myasthenia gravis. W. L.
Nastuk, K. E. OsseRMAN* Anp O. J. PLescra.*
Dept. of Physiology, College of Physicians and
Surgeons, Columbia Univ., N.Y.C., Myasthenia
Gravis Clinic and Dept. of Medicine, Mt. Sinai
Hosp., N.Y.C., and Institute of Microbiology,
Rutgers Univ., New Brunswick, N.J.

In 31 serum complement (C’) analyses on sam-
ples from 12 human controls (8 male, 4 female),
the maz. dev. from the mean was +8%. C’ levels
in individuals (male) determined at 1-3 month
intervals showed a maz. dev. from the individual’s
mean of +7%. On two females, C’ conc. was
determined 2-5 days prior to, the second day of,
and 1-2 days following menstrual bleeding. The
max. dev. of single values from the mean for the
individual was +2%. For the male and female
groups the mean value of C’ conc. was identical.

136

C’ determinations were also carried out on 45
patients with established myasthenia gravis.
Anticholinesterase medication received by the
patients was shown to have no effect on the C’
analysis itself, and had no short term (hrs.)
effect on the patient’s C’ level. For each of 22
patients, 3-13 serial determinations were carried
out over a time span ranging from 2} to 9 months.
For 8 patients, 2 C’ analyses were made, and in the
remaining 15 patients a single serum sample was
analyzed. 32 patients from the myasthenia gravis
series showed, at some time, C’ conc. below the
lowest C’ conc. obtained in the control series. If
the lowest level of C’ reached by these patients is
averaged, the value obtained is only 33% of the
mean value for the controls. Survey of the case
histories gives indication that reduction of C’
conc. is related to exacerbation of myasthenia
gravis symptoms. Remission appears to be coupled
with a return of C’ conc. to the control range.

441. Anoretic responses to radiation and their
effect upon altitude tolerance. BERNARD D.
NEewsom* aNnD Donatp J. KimME.LporF. Bio-
logical and Med. Sciences Div., U. S. Naval
Radiological Defense Lab., San Francisco, Calif.
The food consumption of rats, rabbits, mice,

guinea pigs and hamsters was measured for 3 days

following an approximately mid-lethal dose of
x-irradiation to assess the degree of postirradia-
tion anorexia. Seventy-two hours after irradiation
these animals as well as ad libitum fed and food
deprived (72 hr.) nonirradiated animals were
exposed to an altitude tolerance test. The mor-
tality produced was used as the criterion of
altitude tolerance. The altitude exposure selected
for each species was sufficient to produce a mor-
tality response of 50% or greater in nonirradiated
animals during 4 hr. of exposure. Irradiated rab-
bits and rats exhibited a severe decrease in food
consumption which persisted for the 3 days of
observation. Irradiated rabbits had an increased
altitude tolerance similar to that previously
observed in the rat. (NEWsom AND KIMELDORF.

Am. J. Physiol. 173: 390, 1954). When nonir-

radiated rabbits were deprived of food for 72 hr.

prior to altitude exposure the altitude tolerance
was similar to that of the irradiated animal.

While the food consumption was lower during the

3 days following irradiation in mice the effect was

much smaller than that observed for rats and

rabbits. Guinea pigs and hamsters exhibited only a

slight decrease in food consumption with recovery

occurring after 24 hr. The mice, guinea pigs and
hamsters did not exhibit a significant increase in
altitude tolerance 3 days after irradiation. How-
ever, when nonirradiated mice and guinea pigs
were food deprived the altitude tolerance was
significantly increased. These observations provide

FEDERATION PROCEEDINGS

Volume 1§

further evidence that the postirradiation increage
in altitude tolerance is dependent upon the post-
irradiation anorexia.

442. The adrenal in the premature anacephalic
fetus. Joun Nicuots. Dept. of Pathology, Yale
Univ., New Haven, Conn.

It is well documented that the adrenal cortices
of the term anacephalic fetus are atrophic; indeed
not a few autopsy protocols record them ag
being absent. This atrophy is presumed to be due
to the absence of ACTH from the maldeveloped
fetal pituitary. The single publication on the
premature anacephalic fetus (Virchows Arch,
210: 158, 1912) reports that the adrenal cortex ig
normal in the anacephalic of 5 months gestation,
This paper has largely been ignored. The second
case of a premature anacephalic fetus of 5 months
gestation in which the adrenal glands are normal
will be presented. This finding means that up
to the 5th month the adrenal cortex can develop
normally without stimulus of fetal ACTH (or at
least a low titre) and/or that sufficient maternal
ACTH can pass the placental barrier to maintain
normal development. The gland subsequently
undergoes atrophy to the atrophic condition
found in the term anacephalic. Some maternal-
fetal adrenal-pituitary relationships will be dis-
cussed.

443. Cardiac catheter-tip pressure gauge.
Frank W. Nose (introduced by Bert R,
Boone). Lab. of Technical Development, Nail.
Heart Inst., Bethesda, Md.

Intra-cardiac pressures are usually recorded
with a pressure gauge attached to the external end
of a cardiac catheter. The long liquid column
adversely affects the frequency and phase per-
formance of the system. Acceleration artefacts are
encountered, as well as lack of reproducibility
caused by the profound effects of minute gas
bubbles. In view of the distortions caused by the
liquid column, it has long been considered de-
sirable that the pressure gauge be located at the
intra-cardiac end of the catheter. A catheter-tip
gauge based on the principle of a sonic valve is
being developed and tested. Audible sound is
introduced into one lumen of a double lumen
cathether and is propagated down the catheter to
the pressure sensing element at the tip. When
pressure is applied to the sensing element, 4
diaphragm moves inward and reduces the cross-
sectional area of the sound path. This attenuates
the sound emerging from the sensing element.
The emerging sound is conducted back through the
second lumen of the catheter to a receiver. Varia-
tions of pressure on the sensing element produce
corresponding variations in intensity of the
sound reaching the receiver. The receiver con-

rr ora a wt fs fs oo ss

olume If

increase
he post-

ephalic
gy, Yale

cortices
; indeed
hem ag
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ERT R,
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se per-
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Varia-
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March 1956

yerts these ‘sound intensity variations to electric
current variations, which are then recorded on a
strip chart. The advantages of this particular
catheter tip gauge, in addition to the elimination
of the liquid column, are the almost complete
insensitivity to acceleration and the relative
ease of manufacture.

44. Ovarian nucleic acids in normal and
hypothyroid rats following gonadotrophin
administration. M. R. Nocenti* anp J. H.
LeatHEM. Bureau of Biological Research, Rutgers
Univ., New Brunswick, N. J.

The ovarian weight increment following
chorionic or pregnant mare serum gonadotrophin
administration is greater in hypothyroid than in
euthyroid rats. To appraise ovarian biochemical
changes related to this augmentation, pentose
and desoxypentose nucleic acids were estimated in
normal and hypothyroid rats with and without
gonadotrophin. Thirty-day-old rats were fed 0.5%
thiouracil in a 20% casein diet. Following a
10-day prefeeding period, 10 1.v. of chorionic
gonadotrophin were injected subcutaneously daily
for 10 days; animals were autopsied after 5 and
10 injections. Similarly, 5 1.u. of PMS were in-
jected daily for 5 days only. Ten injections of
chorionic gonadotrophin increased ovarian weight
from 12.6 mg to 80.3 mg in euthyroid rats and to
165.6 mg in hypothyroid rats. Five injections of
PMS increased the ovarian weight from 12.6 mg
to 53.1 mg in euthyroid rats and to 108.5 mg in
hypothyroid rats. Ovarian enlargement was char-
acterized by a significant increase in pentose and
desoxypentose nucleic acid synthesis which
paralleled the augmented weight increase. As with
the weight changes, the augmented nucleic acid
synthesis was apparent after 5 injections of PMS,
while 10 injections were required with chorionic
hormone. The data indicate that hypothyroid-
induced augmentation of ovarian weight response
to these gonadotrophins is a true growth repre-
sented by increased cell number and activity.

45. Quantitation of effects of X-radiation
and oxygen poisoning by the electroretino-
gram. W. K. Noe ann N. A. Batty.* Depts.
of Anesthesiology and Radiation Therapy, Roswell
Park Memorial Inst., Buffalo, N. Y.

The electrical potential recorded as the electro-
tetinogram (ERG) from cornea or anterior cham-
ber in response to a light stimulus provides
atool for quantitative evaluation of many phe-
nomena that produce damage to the visual cells.
Without respect to basic mechanisms or cellular
sites of action of the injurious agents, effects
produced may be quantitatively assessed. It will
be shown that for constant stimulus and adapta-
tion, attenuation of the electroretinographic

AMERICAN PHYSIOLOGICAL SOCIETY

137

components (a- and b- wave) is related to the
number of visual cells affected. This relationship
is unique for each mode of action. Therefore, the
same degree of attenuation serves as an indicator
for dose-effect relationships. Reversibility or
irreversibility of cell damage is demonstrated by
the degree of ERG recovery. Irreversible changes
produce two types of histological damage, either
acute visual cell death or slow degeneration
of the sensory organelles. Differential attenuation
of the a-wave indicates prevalence of the second
type. Visual cell effects produced by x-rays in the
rabbit have been studied with respect to massive
doses, latency periods, dose rate, fractionation
and relative biological efficiency. Oxygen toxicity
has been evaluated in the same animal species as
to modifications by circulatory changes, adrenal-
ectomy, xX-radiation and chemical protectors.
(Aided by PHS grant B-812.)

446. Comparative glycemic response of small
mammals to chlorpromazine. Davin Nor-
MAN* AND WiL1i1AM A. Hiestanp. Lab. of Anit-
mal Physiology, Dept. of Biological Sciences,
Purdue Univ., Lafayette, Ind.

Chlorpromazine (Thorazine, Smith, Kline
and French) was injected intraperitoneally into
mice, rats and hamsters, and blood samples were
later withdrawn from the orbital sinus for blood
sugar determination. Mice receiving CPZ at 5
mg/kg showed a progressive increase in blood
sugar from 142.2 to 203.3 mg % in 30 min. which by
3 hr. had reached 282.8 mg %. A gradual decline
then followed until at 5 hr. the level was only
slightly higher than normal. The rat showed
only slight hyperglycemia even with doses reach-
ing 60 mg % which killed approximately 50%
of the rats. The average glycemia level of un-
treated 24-hr., fasted rats was 89 mg % which
rose to a level of 127 mg % in rats receiving 20
mg/kg 3 hr. after injection. The hamster showed
a glycemic response similar to the mouse though
to a lesser degree. The minimum intraperitoneal
dose necessary to produce a significant hyper-
glycemia was 10 mg/kg, or twice the mouse dose.
Fasting 24 hr. lowered the glycemia level of the
hamster from 108 to 96 mg % which rose to 194
mg % following CPZ. CPZ increased the fasting
hyperglycemia of alloxan diabetic mice and
significantly decreased survival of the diabetic
animals. The average 24-hr. fasting blood sugar
in the nontreated diabetic mice was 374 mg %,
which rose to more than 600 mg % 2 hr. after
injection with CPZ. Protection against hypo-
glycemic convulsions produced by insulin (9
u/kg) resulted with CPZ. Thus all of 16 mice
made convulsive by insulin after receiving CPZ
survived, while 11 of 16 mice made convulsive
with insulin but which-received no CPZ died.

138

(Chlorpromazine (Thorazine) was supplied by
Dr. Fellows of Smith, Kline and French Labs.,
Philadelphia.)

447. Succinylcholine paralysis, artificial res-
piration and carbon dioxide causing pro-
longed central respiratory depression. NILs
NorMANN (introduced by H. K. BEEcHER).
Anesthesiology Lab., Jewish Hosp., Washington
Univ., St. Louis.

Cats, paralyzed by a continuous infusion of
succinylcholine, were exposed to increased con-
centrations of carbon dioxide and to artificial
respiration in which inflation pressure and/or
duration was varied. The degree of neuromuscular
block was monitored by recording of muscle
action potentials from the diaphragm and the
intercostal muscles. In closed-chest experiments,
spontaneous respiratory function was measured
in terms of respiratory minute-volume and tidal
air. In open-chest experiments, action potentials
were recorded from the central section of a cut
phrenic nerve and electrical stimuli were applied
to the peripheral part. Magnitude and relative
duration of inflation were recorded as changes
either in tracheal cannula pressure or in chest
volume. The results indicate that during succiny]-
choline paralysis, the inspiratory inhibitory
Hering-Breuer reflex, accentuated by increased
inflation pressure and duration, combines its
effect with that of depressing concentrations of
carbon dioxide. A prolonged depression on this
basis was observed in animals lightly anesthetized
with either urethane, pentobarbital or thiopental.
Under the conditions of these experiments neither
a direct central depressant action of succinyl-
choline nor a prolonged peripheral block was
observed. (Aided by Research Grant B-783 from
Natl. Insts. of Health, PHS.)

448. Age differences in lung compartments
and‘bellows function. ArTHuR H. Norris,*
NatHan W. SHock, Mitton LANDOWNE AND
Josepu A. FALzone, JR.* Section on Gerontology,
Natl. Heart Inst., Bethesda, and Baitimore City
Hosps., Baltimore, Md.

Lung volumes and bellows function were
measured in 135 male subjects evenly distributed
between 20 and 89 years of age and chosen from
the patients, staff and employees of the Baltimore
City Hospitals Infirmary Division. Standard
methods were used, except for a helium washout
method for functional residual capacity (FRC).
Significant (P = <.001) regression on age was
found for total lung capacity (TLC), maximal
breathing capacity (MBC), and vital capacity
(VC) and its subdivisions, while residual volume
(RV) increased significantly and tidal volume
(TiV), at rest, and FRC did not change sig-

FEDERATION PROCEEDINGS

Volume 15

nificantly. Height, weight and surface area
showed significant age decrement. TLC/m!
surface area did not change (—4 cc/m?/yr)
significantly with age, while VC/m? decreased
(—18 cc/m?*/yr.) significantly and RV/m? jp.
creased (+13 cc/m?/yr.) significantly. Thus, the
changes with age consist of a shift in volume from
the mobile spaces to the fixed space. The con-
tribution of TiV and breathing rate to the con-
tinuous, uniform reduction (—.721 1/m?2/yr.) in
MBC was assessed. The 20-29, 30-39, 40-49 year
age groups show MBC reduction because of TiV
reduction (.92, .74, .65 1.) with equal breathing
rates (124 breaths/min.). The 50-59, 60-69, 70-79,
80-89 year age groups show MBC reduction be-
cause of reduced breathing rates (84, 76, 68, 48
breaths/min.) and similar TiV (between .70 and
.80 1.). The age decrement in MBC is accounted
for by lesser augmentation of voluntary breathing
rate in the older subjects.

449. Influence of some intermediates of the
Krebs cycle upon oxygen uptake of de-
veloping wings and legs of the chick embryo,
Wixtor W. Nowinskr AND Mary H. Parr-
RIDGE.* Univ. of Texas Med. Branch, Galveston,
Texas.

Wing and leg buds of chick embryo in earlier
stages grow at closely parallel rates, which deviate
during ontogenesis. This material was used to
study the relation between growth rates and some
intermediates of the Krebs cycle. Oxygen uptake
was measured by the Warburg technique and
pyruvate, cis-aconitate, isocitrate, alpha-keto-
glutarate, succinate and malate were added as
substrates. On the 6th day the wings showed in-
creased Qo, with pyruvate and succinate. In the
wings on the 7th day all intermediates were
active, on the 8th—only isocitrate, ketoglutarate
and malate. In the legs, on the 6th day only
succinate increased the oxygen consumption,
on the 7th day—pyruvate and succinate, and on
the 8th day, no increase with any of the inter-
mediates could be observed. Calculated on the
100 ug DNA basis (Biochim. et Biophys. Acta 11:
1953; Texas Rep. Biol. K Med. 13: 1955) which
corresponds to a population of approximately
12,500,000 cells, a significant increase in oxygen
consumption with succinate and malate was
observed in the 6th day wings, on the 7th day all
intermediates used were active in the wings and
in the legs. On the 8th day, activities with iso-
citrate, succinate and malate were obtained,
whereas in the legs only malate was active. (Sup-
ported by Public Health Grant # C-2296; C4.)

450. Renal responses to cold exposure of large
v. small dogs. WiLti1am C. NuNGESSER (in-
troduced by BENJAMIN DEBoER). Department

olume 15

ce area
TLC/m:
/m?/yr.)
ecreased
‘/m? in.
hus, the
ime from
The con-
the con-
*/yr.) in
-49 year
e of TiV
reathing
9, 70-79,
tion be-
5, 68, 48

.70 and
counted
reathing

| of the
of de-
smbryo,
. Part.
alveston,

n earlier
| deviate
used to
nd some
1 uptake
que and
ha-keto-
dded as
wed in-
. In the
es were
lutarate
ay only
mption,
and on
e inter-
on the
Acta 11:
) which
‘imately
oxygen
te was
day all
ngs and
‘ith iso-
ytained,
e. (Sup-
74.)

of large
ER (in-
partment

March 1956

of Physiology and Pharmacology University of

Norih Dakota School of Medicine, Grand Forks,

North Dakota

Previously we have reported (Federation Proc.
14: 108, 1955) that trained, unanesthetized small
female dogs (8-10 kg) exposed for one hour in a
room cooled to near 0°C, showed a decrease in
elective renal plasma flow, glomerular filtration
rate and urine volume. Skin temperatures of the
foot-pads and trunk also decreased in these
animals. In the present series large female Labra-
dor-type dogs weighing about 25 kg were studied
under similar conditions. The responses of the
large dogs differed from those of the small dogs
in several ways. The glomerular filtration rate
usually increased, while the renal plasma flow
either increased or did not change. The large dogs
showed a greater fall in trunk surface tempera-
ture than did the small dogs when exposed to the
same cold conditions. The urine volume usually
decreased in both groups. Urine osmolarity and
the osmotic U/P ratio increased, suggesting that
active tubular reabsorption of water was in-
creased in the cold. (Aided by a contract with the
Arctic Aeromedical Laboratories.)

41. Cardiac arrest in the heart-lung prepara-
tion from hyperkalemia. L. J. O’Brien,*
H. W. Diserens,* G. T. Stevens* and M. M.
Guest. Carter Physiology Lab., Univ. of Texas
Med. Branch, Galveston.

Dog heart-lung preparations were used to
study effects of varying the concentration of
potassium on the electrocardiogram and cardiac
function. Heparinized blood was passed through
an artificial circuit slightly modified from the
original system devised by Starling. Potassium
was added as 2% KCl in Ringer’s solution. Analy-
ses of circulating blood at frequent intervals
throughout the experiment included hematocrit,
potassium, sodium and_ glucose. Potassium,
sodium and glycogen in the myocardium were also
determined at the termination of the experiment.
The usually described electrocardiographic
changes occurred as the potassium was increased,
but cardiac arrest regularly occurred when the
concentration reached 7-10.5 mEq/1. if the prepara-
tion was allowed to function at this level for
fom 15-30 min. This is a considerably lower
potassium concentration resulting in arrest than
has previously been reported. In studies in which
the intact animal has been used to determine the
lethal potassium level, the concentration may
not have been maintained at any one level for a
sufficient length of time to determine the ultimate
eect on the heart. Thus, the toxic effects of
hyperkalemia appear to be dependent not only
o the absolute concentration of potassium in
Plasma but also upon the time period during

AMERICAN PHYSIOLOGICAL SOCIETY

139

which the myocardium is subjected to a par-
ticular concentration of this ion.

452. Mechanism of potassium excretion in the
chicken. Jack Or.Lorr AND Dovuge.ias G.
Davinson.* Natl. Heart Inst., Bethesda, Md.
The renal-portal circulatien of the chicken

allows for unilateral peritubular perfusion of test

substances in concentrations otherwise lethal
for the animal. Material injected into a leg vein
enters the peritubular circulation of that side,
initially bypasses the glomerulus, enters systemic
blood and is filtered in equal concentrations
bilaterally. Unilateral urine collections permit
comparison of the experimental kidney with a
contralateral control. Utilizing this preparation,
data have been obtained with respect to K* which
have been interpreted to satisfy 3 criteria for the
presence of a carrier or enzymatic process. 1) The
mechanism for K* secretion is capable of satura-
tion. Maximal rates of K* excretion of one kidney
vary between 60 and 85 um/min. in +2.0 kg chick-
ens. The calculated peritubular plasma concen-
tration necessary to achieve this is approxi-
mately 8.5-10.0 um/ml. 2) peritubular elevation
of pCO2 without concomitant changes in peripheral
arterial pCO. by injection of acetic acid into
the leg vein results in a fall in K* excretion and

a rise in H* excretion. Alternatively, NaOH by

reducing pCO: locally, enhances Kt excretion

when initially low and depresses acidification.

Thus competitive inhibition of a secretory process

has been demonstrated. 3) Reversible, non-com-

petitive inhibition of the process by mercury
results in virtual disappearance of K+ from the
urine when initial excretion is 3-5 um/min. This
may indicate that all filtered Kt is reabsorbed.

That the decrement in K* excretion may exceed

the amount filtered indicates that mercury de-

presses a secretory process.

453. Pulmonary gas exchange in hypothermia.
A. B. Otts, James R. JupE* anp RoLanpD
Fouse.* Dept. of Surgery, Johns Hopkins Univ.,
Baltimore, Md.

End tidal Pco, and arterial Pco, were measured
in dogs at various body temperatures down to
16°C. No increase in the arterial-alveolar gradient
was found, and it is concluded that hypothermia
introduces no physiologically significant barrier to
the transfer of carbon dioxide between blood
and lungs. Measurement of the diffusing capacity
of the lungs has been measured by the steady
state carbon monoxide method in 4 dogs at body
temperatures of 37° and 25°. The measured de-
crease in Dco at the lower temperature is greater
than we would predict on purely physical grounds.
We suggest that a major factor involved may
be a reduction in area of pulmonary vascular bed

140

available for diffusion. This hypothesis is sup-
ported by measurements on 2 of the dogs both of
which showed an increase in pulmonary vascular
resistance at the lower temperature. (Supported
by Contract AF 41(657)-30 with the USAF School
of Aviation Medicine, Randolph Field.)

454. Unusual form of protein-bound serum
iodine in Hashimoto’s thyroiditis. CHARLES
A. OwrEn, Jr. AND WiLiiam M. McConauey.*
Mayo Clinic, Rochester, Minn.

In many patients with Hashimoto’s thyroiditis,
despite a reduced basal metabolic rate, the serum
protein-bound iodine (PBI) is normal or above
normal and the thyroidal uptake of radioiodine
is in the ‘hyperthyroid’ range. Analyses of serums
from such patients revealed that as much as half
of the PBI was not extractable with acidified
butanol and, therefore, was not thyroxine bound
to alphaglobulin in the usual way. After ad-
ministration of 300-500 ue of radioiodine, there
appeared in the blood of these patients protein-
bound radioiodine that was both soluble and
insoluble in acidified butanol. Other situations in
which a large fraction of the PBI is insoluble in
acidified butanol are after prolonged administra-
tion of Lugol’s solution (Man and associates)
and after large doses of I'*! in the treatment of
thyroidal malignant lesions (Robins). In the
latter, one might assume the release into the blood
of thyroglobulin from the disintegrating thyroid
gland. By analogy, the pathologic process of
Hashimoto’s thyroiditis also may lead to the
release of thyroglobulin, rather than thyroxine,
directly into the blood. Keiselguhr column chro-
matography of trypsin-hydrolyzed glands of
patients with Hashimoto’s thyroiditis who have
received I!*! has revealed a fairly normal distribu-
tion of radioactive thyroxine, triiodothyronine,
iodide, diiodotyrosine and moniodotyrosine.

455. Mineral requirements of mammalian
cells grown in agitated fluid medium.
Ouea V. H. Owens,* Marcaret K. Gry* anp
GerorceE O. Gey. Dept. of Surgery, Johns Hopkins
Med. School, Baltimore, Md.

The effect of variations in calcium and potas-
sium concentration on free cells grown in agitated
fluid medium was studied. The culture medium
consisted of human placental cord serum 50%,
bovine embryo extract 10%, and balanced salt
solution 40%. Reduction of cation concentration
was accomplished with an ion exchange resin.
The salt solutions were made up to values similar
to normal serum except for the ion under study.
Mineral determinations were made with a flame
photometer. Altered mouse tumorous lympho-
blasts (strain MB III, de Bruyn-Gey) were used in
studies carried out with roller tubes and by

FEDERATION PROCEEDINGS

Volume 1§

our original technic for growth of free cells in
agitated fluid. By this method, cell counts could
be used to determine growth rate. It was found
that over a wide range (1-10 mEq/1.) calcium ig
not a critical factor in early growth response of
these cells. However, below one fifth of the normal
total calcium value in serum there was a definite
fall in growth rate. Similar studies were carried
out with potassium, covering a wide range above
and below normal level in serum. Increases of the
order of 5 times normal value resulted in pro-
gressively increased growth rates. Beyond this
high level, there was a drop in growth rate,
Progressive decreases to a very low level resulted
in progressive decrease in growth rate. Over the
ranges of calcium and potassium studied here,
there were no dramatic cytological changes noted
during the short period that the cells were under
observation.

456. Diffusion of water into human red cells
in vitro. C. V. PAGANELLI AND A. K. Sonomon
(introduced by Joun R. PAPPENHEIMER),
Biophysical Lab., Harvard Med. School, Boston,
Mass.

A flow system has been devised to measure the
diffusion of water into human red cells in vitro
using titrated water as a tracer. The two jet
mixing chamber is modified from the designs of
Hartridge and Roughton (Proc. Roy. Soc. A 104:
376, 1923) and Chance (J. Frank. Inst. 229: 445,
1940). Freshly drawn, heparinized, undiluted
human blood, or fresh red cell suspensions enter
the chamber through one jet, and titrated red
cell buffer enters through the other. Both streams
are propelled by gas (5% COs, 95% air) at pres-
sures of 15-40 lb/in.? so that the mixed stream
travels at velocities of 5-10 m/sec. Observations
are made by forcing a small amount of extra-
cellular fluid through a ‘millipore’ filter (pore
diameter 0.8 ») placed tangentially to the direction
of flow. The observation port is placed from 2 to5
em from the point of mixing, which allows for
observations 2-10 msec. after diffusion begins.
The diameter of the observation port is 0.15 em,
which introduces an uncertainty of 0.15-0.3 msec
into the time measurements. There is no evidence
of hemolysis of the blood after passage through the
system. Preliminary measurements have been
made on two separate samples of blood leading to
values of 2.9 and 4.0 msec. for the half-time for
diffusion of water into human red cells under our
experimental conditions.

457. Effects of testosterone on removal of
dye-labeled protein from the circulation.
Rita L. Patprno* anp CHESTER Hyman. Dept.
of Physiology, Univ. of Southern California,
Los Angeles.

-_ 3
=

lume 16

cells in
ts could
s found
cium is
onse of
: normal
definite
carried
e above
8 of the
in pro-
nd. this
th rate.
resulted
ver the
d_ here,
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re under

ed cells
}OLOMON
(EIMER),

Boston,

sure the

two jet
signs of
. A 104:
29: 445,
ndiluted
ns enter
ted red
streams
at pres-
| stream
rvations
f extra-
ar (pore
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m 2 to5
lows for

begins.
0.15 cm,
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>vidence
ough the
ve been
ading to
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nder our

oval of
ilation.
n. Dept.
lifornia,

March 1956 AMERICAN PHYSIOLOGICAL SOCIETY 141

An earlier<report from this laboratory showed
that although the removal of reproductive organs
fom female rabbits did not affect the removal
of dye-labeled protein from the circulation, it did
interfere with blockade of the RES by Thorotrast.
It was also reported that the administration of
estrogen increases the dye-removal rate and
restores blockade of the RES by Thorotrast, only
in animals which have uteri. The possibility
that testosterone might influence these functions
was explored in animal preparations in which
either the ovaries or uteri or testes were removed.
In each case the difference between the control
dye-removal rate and the rate measured after
the administration of Thorotrast was determined.
These differences in rates, expressed as per cent
per minute, were:

Female Ovex Hystex Male Castr.
Control 0.21 0.03 0.013 0.024 0.19
Testosterone 0.01 0.08 0.00 0.00

We conclude that there are 3 conditions under
which blockade of the RES cannot be achieved:
1) absence of uterine tissue; 2) absence of ovaries;

| and 8) presence of testosterone. (Supported by a

| grant from the Natl. Insts. of Health, PHS.)
in vitro |
eal | 458. Effect of sex hormones on fat deficiency

syndrome. T. C. Panos,* G. Kuzin,* R. L.
Wauu* anv J. C. Finerry. Depts. of Pediatrics
and Anatomy, Univ. of Texas Med. Branch,
Galveston.

Fat-deficiency signs, as manifested by der-

| matitis and growth retardation, occur earlier and

are more pronounced in growing male than in
growing female rats. Methyl linoleate require-
ments are approximately 5 times greater for male
than for female rats. In order to explore the
causal relationship of gonadal hormones to
these differences, the following experimental
groups, utilizing 194 weanling Holtzman rats,
were studied for 9 wk.: 1) fat-free females; 2)
fat-free ovariectomized; 3) fat-free ovariectomized
and injected with estrone in aqueous suspension,
2ug daily; 4) fat-free ovariectomized and in-
jected with aqueous testosterone, 100 ug daily;
5) fat-free males; 6) chow (Purina) males. Basal
oxygen consumption was determined weekly in
$8 animals from each group. It was character-
istically and equally elevated in all fat-deficient
groups, and thus not affected by difference in sex,
wariectomy or gonadal hormone treatment.
Dermatitis appeared earliest (within 4 wk.) in
hormone-treated animals and became progres-
tively more severe, particularly among those
leceiving estrone. Mild dermatitis appeared after
5wk. in a small percentage of the ovariectomized
fat-free females and the fat-free males. Skin
changes were not observed at any time during

the 9 wk. in the intact fat-free females or chow
animals. Typical growth retardation occurred in
all fat-free animals, but was definitely less pro-
nounced in ovariectomized and in androgen
treated ovariectomized rats, and more pro-
nounced in those receiving estrone. (Supported in
part by Grant A-380 from Natl. Insts. of Health,
PHS.)

459. Effects of adrenochrome on oxidative
phosphorylation in liver mitochondria.
JANE Harting Park, BLANCHE PITTMAN
MERIWETHER AND C. R. Park (introduced by
Frank R. Buoop). Dept. of Physiology, Vander-
bilt Univ. Med. School, Nashville, Tenn.
Adrenochrome (5 X 10-4 m) has been found to

uncouple completely the oxidative phosphoryla-
tion in hamster liver mitochondria using beta
hydroxybutyrate as substrate (P/O ratio 2.2 to
2.6 in controls). Under the same conditions, 1 or
2 X 10-4 m thyroxin reduces the P/O ratio to
zero, and 5 X 10-3 m epinephrine lowers the P/O
ratio by 0.7 units. Adrenochrome~(0.2 to 2 X
10-4 m) also causes an increase in oxygen con-
sumption which varies in the different mito-
chondrial preparations from 20 to 40%. In con-
trast to thyroxin, the uncoupling by adreno-
chrome has not as yet been shown to be signifi-
cantly reversed by increasing magnesium ions
over a concentration range from 0.0067 m to .067
mM. Adrenochrome is a more effective uncoupling
agent in mitochondria in the presence of thyroxin.
At a thyroxin concentration of 5 X 10-° m, which
is not sufficient to affect the P/O ratio, adreno-
chrome (1.9 X 1075 m) will reduce the P/O from
2.5 to 1.2. This concentration of adrenochrome is
about 10 times less than the amount needed to
cause a similar drop in P/O in untreated mito-
chondria and has no effect on phosphorylation
in untreated mitochondria. These effects of
adrenochrome may be related to the marked
stimulation of oxygen consumption in normal
animals and highly sensitive thyrotoxic patients
following secretion or administration of epi-
nephrine.

460. Pregnancy in partially hepatectomized
rats. K. E. Pascuxis, A. CANTAROW AND
J. Stasney.* Div. of Endocrine and Cancer Re-
search, Jefferson Med. College, Philadelphia, Pa.
We have previously reported experiments on

the growth of transplanted tumors in the presence

of liver regeneration following partial hepatec-
tomy. It appeared desirable to investigate the
interrelations of liver regeneration and pregnancy
in the rat. Partial hepatectomy was performed

a) on the 8th day, or b) on the 17th day of preg-

nancy. Control groups consisted of 1) untreated

pregnant animals, 2) nonpregnant hepatectomized

142

rats, 3) sham-operated pregnancy controls, in
whom a laparotomy was performed. All pregnant
animals were killed on the 20th day of pregnancy,
together with the nonpregnant controls. Fetal
damage was severe in the partially hepatectomized
animals; in many, several or all fetuses were
resorbed. The mean number of fetuses recovered
on the 20th day was 9.8 in the pregnancy controls
as well as in the sham-operated pregnancy con-
trols, as against 3.7 in the 8th day hepatectomy,
and 5 in the 17th day hepatectomy group. The
mean fetal weight was 2.1 gm in both control
groups, as against 1.75 and 1.6 in the hepatecto-
mized groups. Conversely, liver regeneration was
identical in nonpregnant and pregnant animals.
Fetal salvage by treatment with estrogen plus
progesterone has been reported in pregnant
animals fed a protein-free diet (NELSON et al.
Endocrinology 55: 453, 1954). However, estrogen-
progesterone administered to pregnant hepatec-
tomized rats was without beneficial effect on fetal
survival or fetal growth. The deleterious influ-
ence of regenerating liver on pregnancy differs
markedly from the influence on pregnancy of
another actively growing tissue, namely, trans-
planted tumors.

461. Effects of serial temporal and parietal
upon discriminative learning in
macaques. Prepro Pasik,* TauBa Pasik,*
WiiuiaMm 8. Barrerspy* anp Morris B. BEen-
pER. Dept. of Neurology, Mt. Sinai Hosp..,

New York City.

Recent studies have shown that bilateral aspira-
tions of temporal cortex disrupt retention on
visual form discriminations. Similar lesions of
parieto-temporal areas, in contrast, have been
said to interfere with tactual performance. So
far, however, the effects of each of these lesions
upon both visual and tactual tasks have not been
evaluated. In the present study, bilateral aspira-
tions @f the ventro-lateral cortex of the temporal
lobe and similar lesions of the posterior parietal
lobule were serially made in each animal. Analo-
gous visual and tactile discriminations of form
and of intensity (brightness or roughness) were
given before and after each operation. Results:
1) temporal lesions disrupted visual retention for
forms 1 in. high, but did not interfere with bright-
ness or tactual discriminations; 2) these same
animals showed no deficits on visual form dis-
crimination when retested with targets twice as
large; 3) parietal lesions did not produce any
impairment upon either tactual or visual testing;
4) these same animals when retested on more
difficult tactual form discriminations still showed
no deficits, even when the lesions were extended
to include portions of the temporal lobe. It is
concluded that bilateral temporal lesions produce

lesions

FEDERATION PROCEEDINGS

Volume 15

a deficit specific for visual form. This disturbanee
can be best explained in terms of a defect in im.
mediate perception, rather than an impairment
in visual memory attributable to damage of some
hypothetical ‘association’ center. Lesions of the
posterior parietal lobule, alone or in combination
with temporal ablations, do not interfere with
tactual functions under our conditions. (Aided by
Public Health Service Grant * B-174 (C38).)

462. Oxygen deficit in the dog gastrocnemius,
Don C. PEar., Jr., L. D. CARLSON AND W. W,
SHERWOOD (introduced by A. A. Warp, Jr),
Dept. of Physiology and Biophysics, Univ. of
Washington School of Medicine, Seattle.

In muscular exercise, a considerable oxygen
deficit is incurred which cannot be explained by
the accumulation of lactic acid. The hypothesis
that this deficit is due to the depletion of high
energy phosphate in muscle and that the oxygen
consumption is increased by an increase in phos-
phate acceptor was tested in the perfused dog
gastrocnemius. The oxygen deficit was calculated
from change in lactic acid and high energy phos-
phate after 2 min. of contraction. The oxygen
deficit was calculated from the oxygen consump-
tion determined at 5-sec. intervals by measure-
ment of blood flow and arteriovenous difference.
For exercise, the muscle was stimulated at 300
eps for 1-sec. intervals with 1-sec. rest. In 14 ex-
periments, the average values of lactic acid were
14.2 mg % at rest, and 29.2 mg % during exercise,
High energy phosphate at rest was 70.3 mg %
and fell to 56 mg % during stimulation. The cal-
culated deficit was 2.3 ce 02/100 gm muscle, and
the measured deficit was 2.2 ce 02/100 gm muscle,
(Supported in part by the U. S. Air Force under
contract No. AF 18(600)-1467, monitored by the
Alaskan Air Command, Arctic Aeromedical Lab.,
APO 731, % Postmaster, Seattle, Wash.)

463. Cardiodynamic responses to electrical
stimulation of the brain stem. CLARENCE N,

Preiss, R. T. Mippo,* Water C. RANDALL |

AND Davin S. Jones.* Dept. of Physiology,
Stritch School of Medicine, Loyola Univ., Chicago,
Til.

A previous report from this laboratory (Ro#s#
AND RanpALL, Federation Proc. 14: 128, 1955)
has presented evidence for a direct effect of elec-
trical stimulation of the sympathetic nervous
system on the force of myocardial contraction.
The present report is an extension of this work
to an investigation of areas in the lower brain
stem which may be involved in such responses of
the heart to sympathetic stimulation. Stimulation
by means of concentric bipolar electrodes has
been carried out in the medulla of cats, using 4
stereotaxic instrument for electrode placement.

“olume 16

turbance
ct in im-
pairment
> of some
1s of the
ibination
ere with
Aided by
38).)

nemius,
ip W. W.
RD, JR.).
Univ. of

» oxygen
ained by
pothesis
| of high
e oxygen
in phos-
ised dog
ilculated
gy phos-
> oxygen
onsump-
measure-
ifference,
d at 300
In 14 ex-
cid were
exercise,
3 mg %
The cal-
scle, and
| muscle,
ce under
1 by the
cal Lab.,

ectrical
ENCE N,

RANDALL ©

ystology,
Chicago,

(RoHSE
3, 1955)
; of elec-
nervous
traction.
his work
er brain
yonses of
nulation
des has
using a
rcement.

March 1956

Exact electrode positions are determined histo-
logically. Accurate recording of systolic and di-
astolic blood pressure was achieved with a Sanborn
electromanometer adapted for optical recording.
In all cases, electrocardiograms were made rou-
tinely throughout each experiment, and in some
cases photoelectric plethysmograms were recorded
from the foot pad. Preliminary results indicate
that many blood pressure responses which have
previously been ascribed to activation of the
yasomotor center do not significantly involve
peripheral vasoconstriction. Stimulation of areas
classically described by numerous workers as
vasomotor, elicited several different types of
response. Some responses appear to be purely
vasoconstrictor. Others appear to be cardio-
augmentor, with or without cardio-acceleration,
and still others seem to involve multiple mecha-
nisms. Exploration of the lower brain stem is
currently in progress to determine whether specific
areas exist for each type of response.

44. Pulmonary damage in high oxygen pres-
sure. KENNETH E. PenrRop. Dept. of Physiology
and Pharmacology, Duke Univ. School of Medi-
cine, Durham, N.C.

In laboratory animals severe pulmonary damage
usually results from prolonged exposure to oxygen
under moderate pressure. The extent to which
human lungs are damaged by similar exposures
is unknown. There is reason to believe, in small
animals at least, the lung damage and the CNS
effect produced by oxygen at high pressure (OHP)
have separate etiologies. Evidence has been ob-
tained that the principal factor involved in the
pulmonary pathology is atelectasis which chiefly
results from isolation of areas due to excessive
secretions in the small airways. The processes of
atelectasis can be delayed or partially reversed by
anumber of procedures such as periodic positive
pressure insufflation, intermittent exposure to an
inert gas such as nitrogen or helium, or by induced
positive pressure breathing. (Work done under a
grant by the Office of Naval Research.)

45. Rapid evaluation of basic functions of
lung using recording oximeter and oxygen
analyzer. JoHN F. Perkins, Jr., WiuuiaMm E.
Apams,* JosepH D. Howarp* and Dario B.
Domizi.* Depts. of Physiology and of Surgery,
Univ. of Chicago, Chicago, Til.

The oximeter-O2 analyzer set-up for determin-
ing the relation between arterial O. saturation
and alveolar O2 tension (‘saturation-tension’
curve, PERKINS, ADAMS AND Forges, J. Appl.
Physiol., Jan. 1956) has been modified so as to
Permit carrying out additional tests. Following
standard ventilatory studies, distribution of in-
spired gas is evaluated by means of N2 washout

S wae on

AMERICAN PHYSIOLOGICAL SOCIETY

143

curves obtained by the method described by
Perkins et al. (Federation Proc. 14: 1955) using
recording O, analyzer and alveolar sampler. Semi-
quantitative evaluation of ‘shunt-like’ effect due
to abnormal distribution of gas and blood and
of anatomical right-left shunting is provided by
the resting saturation-tension curve and of dif-
fusion difficulty in terms of shift of the curve
during exercise. Fifteen normal subjects and 35
patients with pulmonary disease have been
studied. In 13 with emphysema, the Nz washout
curves had 2 or more distinct components and
the saturation-tension curves were depressed,
markedly in some. Findings of this type sometimes
helped to reverse a previous diagnosis of poly-
cythemia vera. In 1 case possessing huge bilateral
pulmonary cysts, with markedly abnormal wash-
out curve, there was little shunting or diffusion
difficulty. Mild to moderate degrees of diffu-
sion difficulty, with other tests usually normal,
were observed in berylliosis, silicosis, granuloma
or fibrosis of unknown etiology, spontaneous
pneumothorax, pneumonectomy. In a cyanotic
child with mucoviscidosis, the resting curve was
shifted markedly to the right, suggesting severe
limitation of diffusion.

466. Patterns of crossed spinal reflex activity.
Epwarp R. PER. Dept. of Physiology, Upstate
Med. Cir., Syracuse, N.Y.

The effects of an afferent volley in a hindlimb
nerve were tested on contralateral monosynaptic
reflexes in acutely spinal cats. The fiber compo-
nents active in the conditioning volley were
monitored by recording from the nerve, from the
ipsilateral dorsal roots or by the reflexes recorded
from the ipsilateral ventral roots. Conditioning
activity restricted to the largest fibers of cutane-
ous nerve (6-124) resulted in a facilitation of
motorneurons supplying contralateral flexors of
the knee and ankle (dorsiflexion) with a latency of
4-5 msec. and a peak effect in 8-15 msec. Such
volleys were commonly associated with a crossed
ventral root discharge similar to that seen ipsi-
laterally. The effect on contralateral flexors could
be initiated from a number of skin nerves with
different distributions. If the afferents activated
in a cutaneous (or deep) nerve included elements
from the smaller myelinated fibers (2-64), a
powerful facilitation of the crossed extensor (knee
and ankle) motorneurons became evident. The
crossed extensor facilitation started 6-30 msec.
after the stimulus, lasted up to several hundred
msec., and was associated with inhibition of the
antagonist flexors. Stimulation of a purely muscle
nerve, particularly a branch to a muscle acting on
the knee, gave rise to excitability changes of the
motorneurons of its contralateral equivalent. On
occasion, activity in some of the larger myelinated

144

fibers of a muscle nerve (? Group II) resulted in a
crossed inhibition manifest within 3 msec. If the
stimulus strength were increased so as to bring
some of the smaller myelinated muscle afferents
into activity, the pattern was that of crossed
facilitation which did not reach a maximum until
most of the Group III fibers had been recruited.
This latter effect reached a peak in 5-10 msec. and
was associated with reciprocal inhibition of the
antagonist.

467. Excretory effects of Diamox on water-
loaded normal and adrenalectomized rats.
J. H. Pertmurr ano D. A. OLEWwINE.* Dept. of
Physiology, Univ. of North Carolina School of
Medicine, Chapel Hill.

Renal excretion of water, Na* and K* during
5-hr. periods was studied in normal and adrenal-
ectomized male rats after an intraperitoneal in-
jection of water (5% b. wt.) and after the same
water load plus Diamox (25 mg/100 gm). Two
weeks after adrenalectomy the animals were used
at weekly intervals alternating between the 2
regimens. Operated animals were maintained on
saline. Eighteen hours before the injections all
animals were fasted but allowed fluid ad libitum.
Compared with the response after water alone,
Diamox increased the excretion of water in normal
animals about 14 times (79.347.8% of injected
water returned vs. 126.8+7.8%) and in adrenal-
ectomized animals about 4 times (12.9+7.6% vs.
50.1+11.8%). Excretion of Nat and K* increased
for both groups after Diamox as compared with
the response after water. The average total ex-
cretion of these ions after Diamox showed certain
significant differences between the 2 groups: a) less
K* was excreted by the adrenalectomized animals
than by the normals (0.099+-0.032 mEq/100 gm vs
0.283+0.028 mEq; P < 0.01); b) if the adrenal-
ectomized animals drank saline during the fast,
they excreted more Nat than the normals
(0.604-46.220 mEq/100 gm vs. 0.359+0.048; P <
0.01), whereas this value was not significantly
different from the normal if they drank water.
Berliner (Federation Proc. 11: 695, 1952) proposed
that when the exchange of Na* for H* is inhibited,
tubular secretion of K* increases. Since the
adrenalectomized rat excreted less K* than the
normal after Diamox but showed essentially a
normal Na* response, the data suggest that tubu-
lar secretion of K+ may be defective in the animal
without adrenals. (Supported by AMA and Univ.
Research Council grants. Diamox . supplied
through courtesy of Dr. T. H. Maren, American
Cyanamid Co.)

468. Conduction block in peripheral nerve
produced by oxygen at high pressure.
PHANOR L. Perrot, Jr.* anv 8S. N. Stern. Naval
Med. Research Inst., Bethesda, Md.

FEDERATION PROCEEDINGS

Volume 61

Scattered reports describe toxicity of oxygen at
high pressure (OHP) on nerve muscle prepara-
tions, but give no direct evidence of functional im-
pairment of nerve per se. Frog sciatic and cat ulnar
nerves were stimulated continuously (20 pulses/
sec.; voltage supramaximal for alpha fibers) in a
pressure chamber at 13 atm. absolute oxygen and
changes in action potential amplitude studied,
Each nerve’s initial action potential amplitude
was made the same by amplifier adjustment and
conduction block was defined as the disappearance
of the action potential during continuous stimu-
lation with the initial parameters. Complete con-
duction block occurred in all nerves tested. Mean
time for block in frog nerves at 13 atm. Oz was 4.5
hr.; in a 5% CO.-95% O» mixture at 13 atm., it was
3 hr. The block was partially reversible on return
to room pressure if not maintained for more than
a few minutes. The presence or absence of con-
tinuous stimulation did not affect the time course
of conduction block. Cat nerves maintained in
Tyrode’s with no added CO: prior to exposure to
OHP were blocked in 13 atm. Oz in 2} hr. Cat
nerves maintained in Krebs-Henseleit equili-
brated with 5% CO.-95% Oz before exposure to
0.38% CO-99.62% O» at 13 atm. (to maintain an
approximately physiological CO. tension during
exposure) were blocked in 50 min. at 37°C. Re-
covery was poor or not evident. The data indicate
that OHP is directly toxic to peripheral nerve.
Mamallian nerve is more susceptible than frog
nerve.

469. Significance of reflected waves within the
arterial system. L. H. Peterson anp P. H.
Gerst.* Depts. of Physiology and Surgery, Univ.
of Pennsylvania, Philadelphia.

It has been frequently proposed that the de-
formation of the proximal (central) aortic pressure
pulse, as it travels peripherally, is due to its fusion
with reflected waves. The ‘standing wave’ hy-
pothesis is an extension of that concept. There are
theoretical objections to these hypotheses, as well
as alternative explanations for the pulse deforma-
tion. There remained, however, a need for experi-
mental evidence of how identifiably isolated
waves, generated in the periphery of the aorta are
transmitted retrograde toward the heart. Experi-
ments have been conducted with living dogs in
which known, controlled volume pulses have been
introduced into the femoral, iliac and distal aortic
vessels. Such pulses induced the formation of
variously shaped pressure waves. The retrograde
transmission of these waves have been recorded
from their sites of origin to the root of the aorta.
It was found, under all conditions, that these in-
duced waves became progressively damped and
did not reach the aortic root. These results do not

lume §1

ygen at
repara-
nal im-
it ulnar
pulses/
s) ina
en and
tudied.
plitude
nt and
arance
stimu-
te con-
. Mean
was 4.5
, it was
return
re than
of con-
course
ned in
sure to
ir. Cat
equili-
sure to
tain an
during
C. Re-
ndicate
nerve.
in frog

1in the
Pie.
, Univ.

she de-
ressure
s fusion
re’ hy-
ere are
as well
forma-
experi-
solated
rta are
Experi-
jogs in
ve been
| aortic
tion of
rograde
»corded
» aorta.
1ese in-
ed and
; do not

March 1956

support the goncept that reflected waves play a
significant role in arterial pulse wave deformation
and indeed, they make the standing wave hy-
pothesis untenable. The results do imply that the
arterial system functions as a damping transmis-
son line rather than as a resonating chamber, and
further, that frequency redistribution is likely to
play a significant role in pulse wave deformation.
(Supported by the Office of Naval Research.)

40. Effect of hyperthyroidism upon cardiac
metabolism in the dog. Dorotny A. PIATNEK
anp Rosert E. Oxson (introduced by Harwoop
§. Betp1na). Dept. of Biochemistry and Nutri-
tion, Grad. School of Public Health, Univ. of
Pittsburgh, Pittsburgh, Pa.

Hyperthyroidism has been induced in a series of
dogs by feeding U.S.P. thyroid powder at the level
of 500-1000 mg/kg/day or by injecting /-thyroxine
subcutaneously at the level of 0.5-1.0 mg/kg/day.
Weight loss (in the presence of increased caloric
intake), greatly increased oxygen consumption,
nild tachycardia, increased hematocrit, increased
excitability, increased skin and rectal tempera-
ture and increased resistance to barbiturate anes-
thesia have been noted. Studies of cardiac work
ad cardiac metabolism were carried out by car-
diac catheterization before and after induction of
hyperthyroidism. The thyrotoxic dogs showed
marked increases in cardiac output and work,
moderate increases in coronary blood flow and
total myocardial oxygen consumption, and de-
meased myocardial extraction coefficients for
pyruvate, lactate and glucose. Studies of distribu-
tion of high-energy phosphate compounds in the
myocardia of these animals at the time of sacrifice
revealed essentially normal levels of creatine phos-
phate and adenosine-triphosphate despite clinical
ad hemodynamic signs of advanced hyperthy-
nidism. Removal of the parotid salivary glands in
sme of these dogs appeared to hasten the onset
ad to increase the severity of the thyrotoxicosis
insupport of the view of Fawcett and Kirkwood
(Science 120: 547, 1954) that the salivary gland is
important for the deiodination of thyroid hor-
mone. Values for protein-bound iodine ranged
ftom 60-90 ug % in thyroid-treated dogs subjected
to parotidectomy, 8-15 wg % in non-operated
thyroid-treated dogs, and 1-4 ug % in normal
ntrol animals. (Supported in part by the Life
hsurance Medical Research Fund.)

fl. Promotion of glycogen fraction synthesis

by magnesium. W. S. Puatner. Dept. of

Physiology and Pharmacology, Univ. of Missouri

School of Medicine, Columbia.

Rats were injected intraperitoneally with 0.1
«MgSO, solution, (0.66 ml/100 gm of b. wt.), and
were killed after 1 and 4 hr. Control rats were
imilarily injected with 0.1 m NaCl and killed at

AMERICAN PHYSIOLOGICAL SOCIETY

145

the same time intervals. Blood, withdrawn by
heart stab, was analyzed for serum Mg. Heart,
skeletal muscle and liver tissues were analyzed for
TCA soluble and TCA insoluble glycogen frac-
tions. One hour after injection of 0.1 m MgSO,
solution, liver TCA soluble glycogen fraction in-
creased from 382+70.7 to 796+144.8 mg% (P =
0.02). Four hours after injection of MgSO,, heart
TCA insoluble glycogen fraction increased from
110+9.7 to 155+11.6 mg% (P = 0.02) and skeletal
muscle, after 4 hr., showed an increase in the TCA
insoluble glycogen fraction from 107+10.1 to
184+13.3 (P = 0.02) mg%. Serum Mg after 1 hr.
was 5.2 mg% and 1.45 mg% after 4 hr. The data
show a sharp rise in liver TCA soluble glycogen
fraction 1 hr. after Mg injection which is coinci-
dent with the high serum Mg levels. Four hours
later as the serum Mg falls to control levels, the
liver TCA soluble fraction also returns to control
levels. Heart and skeletal muscle TCA insoluble
glycogen fractions reach a peak 4 hr. following
Mg injections, but after the serum Mg has re-
turned to control levels. This implies that the Mg
ion preferentially promotes the synthesis of the
TCA soluble fraction in the liver and TCA in-
soluble fraction in heart and skeletal muscle.
(Supported by grants from Lederle Labs. and
PHS.)

472. Decarboxylation of 3 ,4-dihydroxyphenyl-
alanine (Dopa) and its competitive inhibi-
tion in vivo. RoBERT S. PocgRuNp* AND WILLIAM
G. CuarK. V.A. Ctr. and Dept. of Physiological
Chemistry, Med. Ctr., Univ. of California, Los
Angeles.

Previous investigators have demonstrated pres-
sor responses in various animal species from intra-
venously administered Dopa. The presence of 3,4-
dihydroxyphenethylamine (dopamine) in the urine
indicated Dopa decarboxylase activity in vivo.
Proof that the pressor effects are caused by formed
dopamine as well as various factors influencing
Dopa decarboxylation in vivo will be discussed.
In extending this work to the inhibition of Dopa
decarboxylation in vivo, Dopa was administered
intravenously to pithed cats and blood pressure
responses due to the formed dopamine were deter-
mined. Pressor responses were compared both be-
fore and after intravenously injecting compounds
structurally related to Dopa but lacking the amino
group. These compounds have been found to be
competitive and specific inhibitors of Dopa de-
carboxylase in vitro by Hartman, Akawie and
Clark (J. Biol. Chem. 216: 507, 1955). Although it
was possible to compare inhibitory activity in
vitro to that in vivo qualitatively, it was not pos-
sible to do so quantitatively. The relative activity
of each compound was calculated, using 5-(3-
hydroxycinnamoy])-salicylic acid as the standard

146 FEDERATION
reference inhibitor, by comparing the dosages pro-
ducing 50% inhibition of blood pressure response
to Dopa. Of a total of about 35 compounds, 7 were
studied by this procedure, while the inhibitory
activity of the others was determined at an arbi-
trarily chosen dose. Some factors influencing the
degree and duration of inhibition by various com-
pounds will be discussed. (Supported by the Los
Angeles County Heart Assoc. and the Life In-
surance Med. Research Fund.)

473. Streptomycin and adaptive enzyme for-
mation. W. J. PoLGuassz (introduced by EpGar
C. Buack). Dept. of Biochemistry, Univ. of
British Columbia, Vancouver.

The adaptive formation of enzymes for the oxi-
dation of L-arabinose, and for the hydrolysis of
lactose was inhibited in resting cell suspensions of
streptomycin-susceptible E. coli but not in the
resistant and dependent variants. When strepto-
mycin-dependent £. coli was grown on a minimal
concentration of antibiotic, the production of
adaptive enzymes by resting cells was markedly
stimulated by the addition of streptomycin. In the
formation of beta-galactosidase it was possible to
demonstrate effects with streptomycin at concen-
trations which affect cell multiplication. Dihydro-
streptomycin and mannosidostreptomycin were
similar to streptomycin in their effect on beta-
galactosidase formation. The effect of streptidine
on beta-galactosidase formation was negligible
and dideguanyldihydrostreptomycin was without
effect. The effect of streptomycin on adaptive
enzyme formation satisfies the following criteria:
1) antibiotically active forms of streptomycin
affect adaptation but antibiotically inactive modi-
fications do not; 2) susceptible, resistant and de-
pendent forms of the organism respond consist-
ently; 3) the concentration of antibiotic required
to produce an effect on adaptation is equivalent to
the copcentrat ion which affects cell multiplication.

474. Influence of hypothalamico-hypophyseal
portal vessel plasma on ACTH release.
Joun C. Porter anp H. W. RuMSFELD, JR.
(introduced by R. W. Lackey). Depts. of Physi-
ology and Biochemistry, Univ. of Texas-South-
western Med. School, Dallas.

Blood which drained into the sella turcica from
the broken hypophyseal portal vessels of the hepa-
rinized dog following hypophysectomy was col-
lected by aspiration. Plasma proteins from this
blood were separated by low-temperature, alcohol
fractionation followed by starch electrophoresis.
These fractions were assayed for their ACTH-re-
leasing activity using the ascorbic acid depletion
method and were administered intravenously to
hydrocortisone-inhibited, intact rats. ACTH-re-
leasing activity in portal vessel plasma was present
in a globulin sub-fraction. Further purification of

PROCEEDINGS Volume 15
this sub-fraction from 10 ml of original plasm
yielded a fraction containing 1.3 mg of protein
which caused a mean change in adrenal ascorbic
acid concentration of —77 mg/100 gm adrenal
weight. The substance responsible for this ae.
tivity is non-dialyzable, suggesting that it ig q
large protein molecule or is firmly bound to sucha
molecule. It is not ACTH since its activity as
measured by adrenal ascorbic acid depletion jg
abolished by hypophysectomy. Furthermore, this
activity cannot be attributed to epinephrine, nor-
epinephrine, or histamine since injections of large
quantities of these substances into hydrocortisone-
inhibited, intact rats caused no depletion.

175. Influence of concentration on active
transport of sodium and potassium across
the human erythrocyte membrane. Rosegrr
L. Post. Dept. of Physiology, Vanderbilt Univ,
Med. School, Nashville, Tenn.

It is assumed that these ions are at thermody-
namic equilibrium across the membrane when the
internal concentration (per liter of cells) equals
the external (per liter of medium). Under these
conditions net transport is all active. (Even with
a deviation from equilibrium of 20 mEq/I. the
change in net transport was less than 5%.) Net
transport was measured by the changes in the
amounts of ions inside the cells. Glucose and
adenosine were always present as substrate with
phosphate or glycyl-glycine as buffer at pu
7.6+0.3. Cell sodium and potassium contents were
initially adjusted by storage in suitable media at
2°. The transport rate of sodium was approxi-
mately proportional to its concentration in the
range of 1 to 50 mEq/I. with rates ranging from0
to 12 mEq/l. of cells/hr. Sodium transport out-
ward was consistently 1.5 times faster than potas-
sium transport inward provided that the potas-
sium concentration was greater than 10 mEq/l.
This was true even when both rates approached
zero at low sodium concentrations. The ratio of
sodium to potassium rate decreased at higher pa
and vice versa. It was decreased in cells showing
marked hemolysis during sterage. The ratio in-

creased to more than 2.5 at external potassium con- |

centrations of less than 3 mEa/I. and sodium net

transport was also slowed. These results are con- |

sistent with the hypothesis that the active sodium
and potassium transport mechanisms are closely
linked and that active sodium transport is a neces-
sary condition for active potassium transport.

476. Effects of curare and strychnine on com-
ponents of evoked cortical potentials (cat).
Dominick P. PurpurA AND Harry GRUNDFES?
(introduced by Paut F. A. Horrer). Depts. of
Neurological Surgery and Neurology, College of
Physicians and Surgeons, Columbia Univ., New
York City.

st
0)
sti
the
tw
tiv
der
pre
abe
(2-
of

stil
tio
cor
cul
silt
pre
rar
sug
neg
ten

suy
nes
exe
fro’
Dy

Cros
tior
Phy
zon
cyt
sym
The
the

par:
grot
the

lam:
forn
dens
thes
corr
plas
part
are

The
with

whic

olume 15

plasma
protein
ascorbic
adrenal
this ac-
; it isa
0 such 4
ivity as
letion ig
ore, this
ine, nor-
of large
rtisone-

active
L across
Rosert
lt Univ.

ermody-
vhen the
) equals
ler these
ven with
iq/l. the
%.) Net
s in the
‘ose and
ate with
- at pH
nts were
media at
approxi-
n in the
g from 0
ort out-

in potas- |

le potas-
) mEq/l.
proached
ratio of
igher pH
showing
ratio in-

sium con- |

dium net
are con-
e sodium
e closely
3 a neces-
port.

on com-
Is (cat).
-UNDFEST
Depts. of
Yollege of
viv., New

March 1956

Central synaptic inhibitory action of curare or
strychnine permits analysis of certain evoked
cortical potentials. The antidromic response on
stimulating medullary pyramids in unanes-
thetized, succinylcholine-paralyzed cats comprises
two surface-positive spikes and 10-12 msec. nega-
tivity, the latter assigned to invasion of apical
dendrites. This negativity and to a lesser extent its
preceding (second) spike are reversibly reduced or
abolished by intravenous d-tubocuraine-chloride
(2-3 mg/kg). This effect correlates with abolition
of relayed pyramidal tract responses to direct
stimulation at cortical site of antidromic registra-
tion. The surface negative wave evoked by direct
cortical stimulation is affected likewise by single
curare injections. Strychnine (0.3 mg/kg i.v.) acts
similarly, but more profoundly, maximum effects
preceding onset of convulsive bursting, but tempo-
rarily disappearing during the latter. These results
suggest that properties of synapses cannot be
neglected in interpreting the evoked cortical po-
tentials, the negative wave perhaps corresponding
to postsynaptic potential. Strychnine bursting
superimposed on dendritic synaptic irresponsive-
ness suggests ancillary, perhaps anti-inhibitory
exciting origin of bursting. (Supported by grants
from the Donner Foundation, the Muscular
Dystrophy and Cerebral Palsy Associations.)

{i7. Structure of the Pacinian corpuscle. T.
ANDREW QUILLIAM (introduced by JoHn FIELD,
II). School of Medicine, Univ. of California at
Los Angeles, and VA Center, Los Angeles, Calif.
A re-examination of the Pacinian corpuscle from

the mesentery of cats using the electron mi-

croscope has confirmed and extended the observa-

tios made during an earlier optical study (J.

Physiol. 129: 167, 1955). The lamellae of the outer

zone of the corpuscle are extremely thin yet truly

cytoplasmic in nature, and they exhibit a radial
symmetry about the central longitudinal axis.

They are arranged in two well-defined groups in

the more superficial of which the lamellae are com-

paratively widely spaced, while in the deeper
group they lie closer together. Over the surfaces of
the lamellae lie many circularly orientated col-
lagen fibers. A few of these fibers cross the inter-
lamellar spaces which otherwise present a uni-
formly amorphous appearance of low electron
density. A distinct layer of thicker cells separates
these outer lamellae from the inner core of the
corpuscle. This latter consists of twin sets of cyto-
plasmic sheets bilaterally enfolding the terminal
part of the central axon. The two halves of the core
are separated from each other by a radial cleft.

The axon, which is somewhat oval in cross section

with its long axis coincident with that of the cleft,

has a limiting membrane immediately deep to
which is a striking pallisade-like arrangement of

AMERICAN PHYSIOLOGICAL SOCIETY

147

mitochondria. No myelin is distinguished around
the terminal axonal filament, but, proximally,
nearer the point of exit of the axon from the
corpuscle, a myelin sheath is acquired, and here
only a few centrally situated mitochondria are
demonstrable. Vascular capillaries occur in con-
siderable number near the myelinated segment of
the axon but are not found further distally.

478. Dependence of lung mechanical proper-
ties on anatomic relationships within
terminal lung units. Epwarp P. Raprorp, Jr.
AND Martua McLavuGuurn (introduced by
Lucien Brovuna). Dept. of Physiology, Haskell
Lab. for Toxicology and Industrial Medicine, E. I.
du Pont de Nemours and Co., Newark, Del.
Quasi-static pressure-volume characteristics of

excised gas-free rat and dog lungs have been
studied during air inflation with and without
previous saline filling. Simultaneously photo-
graphs of the lung surface have been obtained to
determine the number and dimensions of surface
units. During inflation, after the ‘opening pres-
sure’ was reached, the number of open units in-
creased gradually; their mean diameter during this
phase was 200-300 microns for both species. As
inflation progressed the mean diameter decreased
due to the appearance of alveoli as projections
from the large underlying alveolar sacs. At full
inflation their mean diameter was about 50 and 80
microns for rats and dogs, respectively. The de-
flection pressure-volume curves differed markedly
from inflation, and the dimensions of surface
structures did not correlate closely with pressure
or volume. Their diameter decreased progressively
until at zero pressure the alveoli contained trapped
air bubbles 30 microns in diameter or less. At the
same time the large subsurface units (alveolar
sacs or ducts) disappeared in irregular sequence
until at a pressure of 2 cm of water they had dis-
appeared completely. These results were not mod-
ified by previous addition of saline alone, but
dilute solutions (0.1-0.5%) of cationic and anionic
surface active salts, and nonionic agents of the
Tween series, produced marked alteration of lung
mechanics and the geometry of surface units,
particularly during deflation. These results indi-
cate that surface forces are a primary determi-
nant of the structure of terminal lung units.
The equilibrium of these forces is unstable and
leads to closure of alveoli and other units with
trapping of bubbles; the phenomenon of closure
probably occurs even at normal lung volumes
and may account for crepitant rales or other
breath sounds.

479. Behavior of pneumothorax gas at sea
level and altitude. HERMANN RAHN AND TULIO
VELASQUEZ.* Dept. of Physiology, Univ. of
Rochester School of Medicine and Dentistry,

148

Rochester, N. Y., and Inst. of Andean Biology,

Lima, Peru.

Two hundred and sixty-two O2 and CO: analyses
describing the composition of pneumothorax gas
have been collected from the literature. When
these data are plotted on the O2.-CO, diagram they
appear to follow a curve described by the blood
R.Q. line originating at a normal arterial gas
tension. (Such a blood R.Q. line represents all pos-
sible venous gas tensions that can exist with
changes in perfusion when the arterial concen-
tration is fixed and the blood exchanges at a con-
stant R.Q. in the tissues.) This suggests that the
pneumothorax gas tensions may be nearly in
equilibrium with the tensions of the venous blood
draining the surrounding pleural tissues. If this
hypothesis is correct, then the pneumothorax gas
tensions at altitude should fall upon another,
predictable blood R.Q. line originating at the new
arterial gas tension. Forty gas samples were
analyzed from patients residing in the Sanatorium
of Jauja, Peru, at an altitude of 11,000 ft. These
values scattered closely around the predicted
blood R.Q. line. On the basis of these findings and
the assumption of near-equilibrium between pneu-
mothorax gas and the venous blood, one may
theoretically predict from the gas analysis not
only the minimal perfusion rate of the pleura but
also the rate of gas resorption from the pneumo-
thorax cavity at any altitude. (Supported in part
by the USAF and the Rockefeller Fndn.)

480. Urinary substance from adrenalecto-
mized rats provoking sodium retention in
rats with intact adrenals. ELAINE P. Ratti,
Epvarpo Ort1,* Mary E. DumM anp BERTRAM
LaKEN.* Dept. of Medicine, College of Medicine,
New York Univ.-Bellevue Med. Center, New York
City.

Urines were collected from normal and adrenal-
ectomized rats on diets adequate or restricted in
sodium. The untreated urines were injected into
hydrated intact, adrenalectomized or hypophysec-
tomized rats. The sodium and potassium excretion,
following injection, was determined in timed urine
samples. The urines from sodium-restricted
adrenalectomized rats provoked definite sodium
retention when tested in normal rats (urines col-
lected from 3 groups of rats, assayed 6 times).
Urines from adrenalectomized rats on a high
sodium intake did not cause sodium retention
when injected into intact rats. Sodium excretion
was not affected in adrenalectomized rats injected
with urines which caused sodium retention in in-
tact rats. Sodium retention also occurred when
urines from sodium restricted-adrenalectomized
rats were injected into recently hypophysec-
tomized rats. Four units of commercial ACTH did
not produce sodium retention when injected into

FEDERATION PROCEEDINGS

Volume 15

hypophysectomized rats. The data suggest the
presence of a urinary substance capable of pro.
voking sodium retention in a test animal with jn-
tact adrenals. This substance is not present jp
commercial ACTH. (Aided by a grant from the
Josiah Macy, Jr. Fndn.)

481. Mechanical impedance to pulsatile blood
flow in hind-limb of the dog. J. E. Ranpai*
AND R. W. Sracy. Inst. of Biophysics, Ohio State
Univ., Columbus.

Evidence is accumulating that a plot of pul-
sating blood pressure vs. blood flow describes a
loop rather than the curvilinear line which jg
characteristic for steady pressure conditions. It
has been suggested that under pulsatile conditions
the mechanical factors of compliance, inertance
and rate of change act in addition to friction in
determining pressure-flow relations. In an attempt
to evaluate the role of these factors, the mechani-
cal impedance to pulsatile blood flow was deter-
mined as a function of frequency from the magni-
tude and phase relations between femoral arterial
pressue and flow Fourier sinusoidal components in
the dog. The nature of the impedance indicates
that a mechanical resonance may exist. A simple
physical model is suggested which contains some
of the frequency characteristics of the vascular
system studied. From such a model rough values
of compliance and inertance were computed. (Sup-
ported in part by a research grant from the Natl.
Heart Inst., Natl. Insts. of Health.)

482. Inhibitory interaction in the eye of
Limulus. FLoyp Ratuirr* anp H. K. Harr-
LINE. Rockefeller Inst., New York City.

The frequency of the discharge of impulses in
any one optic nerve fiber from the lateral eye of
Limulus depends not only upon the illumination
on the specific sensory element (ommatidium)
from which that fiber arises, but also upon the ac-
tivity of neigboring elements, which exert in-
hibitory influences upon it. Simultaneous records
of the responses in two optic nerve fibers show that
the decrease in frequency of the discharge from
one sensory element, caused by activity of the
other, increases linearly with the frequency of the
discharge from the latter. Since the response of
each element thus depends in part upon the re-
sponse of the other element in the interacting pair,
the responses of both may be described by a pair of
(linear) simultaneous equations (one equation for
each element) each of which includes a term for the
response produced directly by the stimulus to the
element and a term for the inhibition exerted upon
that element by the other element in the pair. The
responses of each of a number of interacting ele-
ments may be described similarly by a set of simul-
taneous equations. In each of these (linear) equa-
tions, the part representing the inhibition includes

lume 1§

est the
of pro-
vith in-
sent in
‘om the

e blood
NDALL*
to State

of pul-
Tribes a
hich is
ions. It
ditions
ertance
‘tion in
ittempt
echani-
3 deter-
magni-
arterial
rents in
dicates
simple
1s some
ascular
values
1. (Sup-
e Natl.

eye of
Harr-

ilses in
| eye of
ination
tidium)
the ac-
ert in-
records
ow that
re from

of the
y of the
onse of
the re-
ng pair,
. pair of
tion for
for the
s to the
od upon
ir. The
ing ele-
f simul-
*) equa-
neludes

March 1956 AMERICAN PHYSIOLOGICAL SOCIETY 149

the sum of terms representing the inhibitory in-
fluences exerted upon the element in question by
all of the mutually interacting elements in the
population. The activity of single optic nerve fibers
elicited by direct illumination of their ommatidia
and subjected to modification by various patterns
of illumination of nearby sensory elements, has
been described quantitatively by this simple
schema.

483. Adrenocortical steroid C-20 reductase
and A‘-3-ketoreductase of rat liver. R1cHARD
0. RECKNAGEL (introduced by F. Maurtz). Dept.
of Physiology, Western Reserve Univ., Cleveland,
Ohio.

Pyridine nucleotide linked enzyme systems of
rat liver catalyze the reduction of the C-20 ketone
and the A‘-3-ketone of adrenocortical steroids.
With crude homogenates or with the microsome
fraction alone, addition of TPN, Mn**, and iso-
citrate, with or without added isocitric dehy-
drogenase, induces a maximum rate of reduction
of the C-20 ketone. When DPNH was supplied the
rate of the reaction was only one-fifth the maximal
tate. Direct analysis for DPNH by spectrophoto-
metric assay at 340 my established that an added
alcohol dehydrogenase system was able to over-
come the endogenous DPNH oxidase (cytochrome
bs) present in the microsomes. An earlier report of
instability of the C-20 reductase system can now
be attributed to the decay of an endogenous
TPNH generating system, since the C-20 reductase
is stable to hypotonic conditions, freezing and
lyophilization. Following lyophilization, the mi-
crosomes yield an unknown CHCl; extractable
substance with strong U-V absorption peaking at
230 my. This circumstance interferes with study of
4.3-ketone reduction in lyophilized microsome
preparations. Disappearance of adrenocortical
steroids without the 17-hydroxyl group (cor-
ticosterone, desoxycorticosterone) was followed
with the blue tetrazolium reaction. These steroids
react to a much lesser extent than do steroids with
the 17, 21-dihydroxy-20-ketone configuration. Mi-
crosomes were prepared by direct high speed
centrifugation, by precipitation in the presence
of ammonium sulfate (40% saturated, pH 7.1) and
by 0.01 m CaCle. In all cases the microsome frac-
tion contained significant A‘-3-keto reductase ac-
tivity. This activity persisted after several cycles
of resuspension and resedimentation. The conclu-
sion of Tomkins and Isselbacher (J. Am. Chem.
Soc., 76: 3100, 1954) that all A‘-3-keto reductase
activity is soluble cannot be confirmed.

44. Effect of position upon respiratory
minute volume of anesthetized dogs. E. A.
Reep. Dept. of Physiology, Hahnemann Med.
College, Philadelphia, Pa.

Respiration was recorded by a Peck-Waller tidal
volume recorder in dogs anesthetized with Nem-
butal. The average respiratory minute volume,
tidal volume and rate vary with the position of
the dog. When the supine, horizontal dog is tilted
head downward 30°, the minute volume and rate
increase while the tidal volume decreases. When
tilted head upward, the reverse occurs. Immedi-
ately after the change in position, the change
in each of these functions is most marked. This is
followed by a return toward, but not to, the pre-
vious value. The initial changes and subsequent
establishment of new equilibria are to be explained
in terms of the Hering-Breuer reflex and pCOs.

485. Endocrine influences upon uptake of
radioactive colloidal gold (AU) by
reticulo-endothelial organs. SHERWwoop M.
RericHarp,* ABRAHAM EDELMANN AND ALBERT
S. Gorvon. Depis. of Biology, Grad. School of
Arts and Science, New York Univ., New York
City, and Brookhaven Natl. Lab., Upton, N. Y.
Adrenalectomy caused a significant increase in

uptake of Au!’ by spleen and liver. This occurred

as early as 30 min. and was maintained for at least

24 hr. The lung, lymph nodes and thymus accepted

small quantities of Au'®, but uptake by these

organs seemed unaffected by adrenalectomy.

Replacement treatment in the adrenalectomized

rat with lipo-adrenal extract, cortisone, hydroxy-

corticosterone, corticosterone or a combination of

DCA and hydroxycorticosterone reduced the

Au’ concentration in the liver to normal levels;

no significant alterations were noted with DCA

alone. Only a combination of DCA and hydroxy-
corticosterone restored the concentrations of

Au! in the spleen to normal. Hypophysectomy

augmented the uptake of Au! by the spleen, liver

and lung as early as 30 min. after its administra-
tion. The lymph nodes and thymus of hypophy-
sectomized animals also accepted larger quantities
of Au! than normal animals. Replacement treat-
ment in the hypophysectomized rat with GH,
ACTH, hydroxycorticosterone, or a combination
of hydroxycorticosterone and GH significantly de-
creased the Au! concentration in the spleen, the
latter 3 hormone preparations lowering the values
to normal. ACTH, hydroxycorticosterone, or
hydroxycorticosterone and GH also significantly
reduced values in the liver below those seen in
operated controls; GH had no effect. Epinephrine
significantly increased uptake of Au! by the
spleen and liver above the levels in adrenalec-
tomized controls, and by the liver and lung over
that in hypophysectomized controls. (Work done
under the auspices of the U. 8S. Atomic Energy
Commission.)

486. Cobaltoproteins of rat liver. Joun M.
REINER AND BuENA REINER.* Simon Baruch

150 FEDERATION PROCEEDINGS Volume §

Research Labs. and Health Research, Inc., Saratoga

Springs, N.Y.

The biochemical function of Vitamin By is
not too well understood, nor is it certain that this
is the sole active form of cobalt in the cell. As a
preliminary to an enzymatic approach to the
problem, the cobalt of the rat has been labeled
with a single dose of radioactive cobalt. The
tissues of the animal were then subjected to differ-
ential centrifugation to separate particulate frac-
tions from one another and from soluble proteins.
The proteins have been subjected to further frac-
tionation by the usual methods. The fractionation
has been followed by assay of radioactivity and of
protein content. The fractions with highest
activity:protein ratio were selected for further
study and subfractionation. The degree of puri-
fication has been followed electrophoretically.
The most active cobaltoproteins have been
subjected to various methods of resolution,
in conjunction with spectrophotometric and
chromatographic techniques, to ascertain the form
of the bound cobalt. Protein-bound cobalt is
widely distributed in the liver cell, and an ap-
preciable proportion of it appears to be in some
other form than that of Vitamin Bi2. (Supported
by a grant from the Div. of Biology and Medicine,
Atomic Energy Commission.)

487. Limitation of experimental congestive
edema by self-selection of fluid intake.
W. O. Rernuarpt. Dept. of Anatomy, Univ. of
California, Berkeley.

Acute massive congestive edema can be pro-
duced in the rat by ligation of the inferior vena
cava (between the liver and right renal vein) if
the animal is given free access to 1% NaCl (Proc.
Soc. Exper. Biol. & Med. 78: 335, 1951). The present
experiments were designed to determine if such
animals would become edematous when allowed
free cheice of water or saline, as in the self-selec-
tion experiments of Richter. Operated rats were
given access to water, 1% NaCl, or choice of either,
and were weighed twice daily until subsidence of
edema. Ten rats given access to saline gained 20%
of their body weight, on the average, as compared
to —1% for rats given access to water. Thirty
rats given access to water or saline gained 6%
during the period. Of the last group, 2 animals
gained 20 and 31% whereas 2 animals in the saline
group did not increase their body weights. The
data are interpreted as indicating that under
conditions leading to edema formation, rats
appear to select an intake which will limit such
edema. Figures for the fluid intake of rats given
self-selection of fluid intake showed a relatively
high early intake of saline, but with a progressive
increase in the intake of water during the experi-

mental period. (Assisted by a grant from the
Natl. Insts. of Health.)

488. Effect of Diamox on bicarbonate exere.
tion during metabolic acidosis. ARNOLD» §,
ReELMAN, G. JAMES ToBIAS AND WILLIAM B,
Scuwartz (introduced by A. H. Heanausr),
Depts. of Medicine, Boston Univ. School of Medi-
cine and Tufts Univ. School of Medicine, Boston,
Mass.

The effects of 10-40 mg/kg of Diamox have been
studied in 25 experiments on dogs with varying
degrees of metabolic acidosis. Plasma bicarbonate
was lowered to 4-20 mEq/l. by prior administra-
tion of ammonium chloride or hydrochloric acid,
In every experiment administration of the car-
bonic anhydrase inhibitor produced an un-
equivocal rise in urine pH and _ bicarbonate
excretion. The effect was largest at the higher
plasma bicarbonate concentrations and tended
to diminish at lower concentrations. The reduction
in bicarbonate reabsorption (calculated per 100 ce
GFR) produced by Diamox was proportional to
the plasma level, but a significant effect could still
be demonstrated at concentrations of 4-6 mEq/l,
These data demonstrate that there is no ‘floor’
for bicarbonate reabsorption below which this
process is totally independent of carbonic an-
hydrase activity. During severe metabolic
acidosis, the inhibitor is effective at filtered loads
which are below the residual rate of bicarbonate
reabsorption which continues after administration
of the inhibitor to non-acidotic dogs.

489. Potentiation of auditory cortical re-
sponses to medial geniculate stimulation.
GIANFRANCO Ricct AND WALTER ROSENBLITH
(introduced by Ciara M. Szeco). Univ. of
California at Los Angeles, VA Hosp., Long
Beach, Calif., and Massachusetts Inst. of Tech-
nology, Cambridge.

Auditory cortical responses to medial geniculate
stimulation were recorded in cats immobilized
with tubocurarine, flaxedil or midbrain tegmental
lesions. The response to a single shock usually
consisted of a positive wave followed by a negative
wave, with respective peak latencies of 2-5 and
5-10 msec. The amplitude and peak latency of
both components were increased with repetitive
stimulation, rates of 10-20/sec. being most effee-
tive. Repetitive stimulation of the auditory radia-
tions and occasionally repetitive click stimuli
gave similar response augmentation. Local cortical
application of Nembutal or veratrine prevented
augmentation of the late negative component,
while strychnine enhanced it. Repetitive stimula-
tion of appropriate thalamic relay nuclei gave
analogous augmentation of responses in the visual
and somatic sensory cortex.

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March 1956

190. Effect of carbon dioxide exposure on
adrenal 17-hydroxycorticosteroid secretion
in dogs. JouHN B. Ricuarps Anp 8. N. STEIN
(introduced by T. L. Wiuumon). Naval Med.
Research Inst., Bethesda, Md.

Male mongrel dogs with polyethylene cannulas
in the right lumbo-adrenal vein and carotid artery
were placed in a recompression chamber at at-
mospheric pressure. Oxygen and carbon dioxide
content of the chamber were measured by con-
tinuous sampling through a Beckman oxygen
analyzer and a Liston-Becker infrared carbon
dioxide analyzer. Following a control period of 30
min., during which adrenal venous and arterial
blood samples were collected, the dogs were sub-
jected to various concentrations of carbon dioxide
inair. Experiments were divided into the following
groups: unanesthetized dogs exposed to 2.5%,
5.0% and 10% carbon dioxide for periods of 1 hr.
each; anesthetized dogs exposed to 2.5%, 5.0% and
10% carbon dioxide for 1 hr. each, 10%, 20% and
30% carbon dioxide for 1 hr. each, 20% carbon
dioxide for 4 hr. and anesthetized hypophysec-
tomized dogs exposed to 20% carbon dioxide for 3
hr. Intermittent arterial and adrenal venous blood
samples were obtained during the carbon dioxide
exposure periods. Adrenal venous bloods were
analyzed for 17-hydroxycorticosteroids. Arterial
bloods were collected under oil and analyzed for
pH, carbon dioxide content, and in some cases
oxygen content. Adrenocortical stimulation was
correlated with decreased pu and increased carbon
dioxide content in the arteria] blood. Maximal ad-
renocortical stimulation occurred in all dogs
exposed to 20% carbon dioxide and this response
persisted for as long as 4 hr. Hypophysectomy
ibolished the adrenocortical response to carbon
dioxide exposure.

41. Skin response of albino mouse to ultra-
violet radiation and photorecovery. ALVIN
F, Rieck AND Eve Carou Rupticu (introduced
by A. K. Parpart). Dept. of Physiology, Mar-
quette Univ. School of Medicine, Milwaukee, Wis.
Young male albino mice were exposed to ultra-

violet wavelengths 0.313 u and shorter 5 days each

week. Doses of the magnitude of 5.55 X 10’

etgs/em! resulted in the death of many mice

during the first 15-20 days of the radiation period.

However, the survival curves showed that the

death rate was increased when animals were kept

in darkness between each ultraviolet exposure,
vhen compared to animals which were kept in
visible light after the same radiation. Following
this early period of rapid death rate the survival
curves leveled off about 16-20 days after the be-
ginning of the radiation period. This phenomenon
weurred at an earlier time but at a lower survival

AMERICAN PHYSIOLOGICAL SOCIETY

151

level when animals were kept in darkness than
when kept in light between each exposure. A
sequential study of the skin on the ears of the
mice was made to study the response of the skin
under various experimental conditions. At the end
of 16 exposures the epidermis on the ears of those
mice kept in darkness was about 4 times as thick
as the epidermis of such animals which were kept
in light. Whereas, the epidermis on the ears of
animals kept in light was about twice as thick as
the nonirradiated control epidermis. These find-
ings indicate that the hyperplastic response of the
epidermis occurs at a more rapid rate when ani-
mals are kept in darkness instead of visible light
intervening each ultraviolet exposure.

492. Serum magnesium and _ hibernation.
M. L. RrepEsEx* ann G. E. Foux, Jr. Dept. of
Occupational Health, Univ. of Pittsburgh, Pitts-
burgh, Pa., and Dept. of Physiology, State Univ.
of Iowa, Iowa City.

The possible contribution of the magnesium ion
to temperature regulation of hibernating animals
was investigated. Serum electrolyte and hemato-
logical data were obtained on bats, 13-lined ground
squirrels and golden hamsters. The serum calcium
(method of Natelson and Penniall) of active and
hibernating animals of the same species was
similar. The serum calcium concentration of the
little brown bat (Myotus lucifugus) showed a
transitory decrease during entrance into and
arousal from hibernation. The potassium deter-
minations by flame photometry showed no sig-
nificant changes due to hibernation. Magnesium
levels (method of Orange and Rhein) were con-
sistently higher during hibernation than in active
animals. The percentage increase in the serum
magnesium varied with the species studied: little
brown bat 62%, big brown bat (Eptesicus fucus)
53%, ground squirrel 65% and golden hamster
25%. The serum specific gravity and blood hema-
tocrit increased slightly with hibernation but
not consistently. The elevation of serum mag-
nesium in little brown bats occurred after only
1-2 hr. of hibernation and was found by the time
the esophageal temperature had dropped to 13°C.
There was no significant serum magnesium in-
crease when oral temperature had dropped to
17°C. Awakening from hibernation does not require
a lowering of the serum magnesium since there was
no reduction of serum magnesium when body
temperature was raised to 18°C. One hour after
arousal from hibernation the serum magnesium
had dropped to the level characteristic of active
animals. (Research aided by Natl. Science Fndn.)

493. Radiation-induced changes in fibrinogen
solutions. PETER RigESsER AND Ropert J.

152

Rutman. Dept. of Zoology, Univ. of Pennsyl-

vania, Philadelphia.

Solutions of purified bovine fibrinogen (px 7.4
and ionic strength of 0.15) were exposed to single
x-ray doses, and the firm-gel time upon the addi-
tion of purified bovine thrombin was determined
at 37.5°C. The clotting time was increased with
increasing X-ray dosage in the ranges of 25-100 r
and 100-500 r. In each case a linear relationship
was obtained between the logarithm of the clotting
time and the dosage. The clotting delay was pro-
portional to the thrombin concentration but
independent of fibrinogen concentration. Addition
to fibrinogen solutions of 10-* m cysteine or glu-
tathione failed to alter the radiation-induced
clotting defect but prolonged the clotting time in
both the irradiated and unirradiated solutions.
Thioglycollic acid (10-* m), as well as higher
concentrations of cysteine (10 m) completely
inhibited the clotting process. The rate of solution
in 5 m urea (final concentration) of fibrin clots
obtained from irradiated fibrinogen was acceler-
ated. Preliminary experiments indicate that the
amount of peptide liberated by the action of
thrombin on irradiated fibrinogen is decreased.
The thrombin clotting time of human and bovine
plasma is not altered by the low doses of irradia-
tion employed, nor is the clotting time of
clotting time of irradiated purified fibrinogen
delayed when thrombin-free human or bovine
serum is added. (Work performed under U.S.
Atomic Energy Commission contract AT (30-1)-
1554.)

494. Rapid procedure for erythrocyte sedi-
mentation and hematocrit studies with
some resulting distribution values in vari-
ous healthy and diseased populations.
V. T. Ritey anp W. C. VauusEs (introduced by
M. B. Zucker). Sloan-Kettering Inst. for Cancer
Resegrch, Div. of Exptl. Chemotherapy, and Volun-
teer Dept., Memorial Ctr. for Cancer and Allied
Diseases, New York City.

The conventional erythrocyte sedimentation
rate and the packed red cell volume procedure has
been modified for mass screening purposes to
effect substantial economies in time and equip-
ment without sacrifice of essential accuracy. The
essence of the modification consists in determining
the sedimentation rates and hematocrit values
directly in the original blood collection tube
without the necessity of the usual transfer to a
secondary, calibrated hematocrit tube. To ac-
commodate the variable volumes of blood
collected and to obviate the necessity of cali-
brated tubes, a proportional volume chart has
been constructed to give the corrected percentage
sedimentation values for the red cells irrespective
of the volume fluctuations and the varying column

FEDERATION PROCEEDINGS

Volume i

heights among the samples. A complementary
scale on the same chart applies similarily to th
packed red cell hematocrit values. As a cong.
quence of the larger diameter tube, the
erythrocyte sedimentation period is reduced from
the standard Wintrobe time of 60 min. to 30 mip,
to yield an approximately equivalent value. h
addition, the centrifugation time for the packed
red cell determination is reduced from the stand.
ard of 30 to 45 min. to a period of 5-10 min. to give
an equivalent packing with the same relative
centrifugal force.

495. Effects of diminishing the oxygen tension
of inspired air on the dilution of labels
intravenously injected. G. C. Ringe anp VW,
G. Moss. Dept. of Physiology, Univ. of Miami
School of Medicine, Coral Gables, Fla.

In dogs there is ample evidence that shunts exist
in the pulmonary circuit. In this report, it will be
shown that moderate lowering of the oxygen in
inspired air modifies the dilution curves of labels
injected intravenously. These changes, we believe,
are those to be expected if shunts in the pulmonary
circuit closed. For example, the appearance time
(AT) should be lengthened when short circuits are
eliminated provided the heart output and central
volume remain unchanged. However, since heart
output and central volume cannot be held con-
stant, one needs to find a procedure which will
correct for such changes. We have developed an
empirical formula PCT/+/AT (PCT = peak con-
centration time) which remains reasonably con-
stant in control observations where layer changes
in heart output and central volume occur. It can
be shown that this ratio falls when anesthetized
dogs breathe air containing 10-15% oxygen. This
is the change to be expected if (AT) is lengthened
and (PCT) does not change. We postulate that
this indicates closing of shunts. Other evidence
(change in peak concentration and shape of rising
limb) also fit the supposition that shunts close
when inspired air contains between 10 and 15%
oxygen.

496. Protein composition of rat uterine lumi-
nal fluid. Ira RrineierR (introduced by
FrEpERICcK L. Hisaw). Biological Labs., Harvani
Univ., Cambridge, Mass.

Accumulation of uterine luminal fluid in the rat
under estrogen stimulation has been well docu-
mented; however, the absence of information on
the chemical nature of this fluid motivated a study
of the protein components. The total nitrogen
and protein concentrations of estradiol-17B,
estrone and estriol induced uterine luminal fluid
are 0.78, 0.40, 0.33 mg/ml and 3.49, 2.36, 2.4
mg/ml, respectively. The estrone and _ estriol
values simulate the concentrations found in
normal proestrous fluid. Analyses of protein by

co
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the
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thy
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E labels
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March 1956

boundary electrophoresis and sedimentation was
conducted on luminal fluid previously concen-
trated by dialysis against clinical dextran or
bovine serum albumin. Six peaks were observed in
estrone luminal fluid with electrophoretic mobili-
ties (barbiturate buffer, px 8.6, u-0.1) which were
significantly different from those of rat plasma.
Six components were also identified by sedimenta-
tion of this fluid as compared to 3 in plasma. The
percentage composition of components obtained
by electrophoresis and sedimentation is also
reported. No lipoprotein was evident in concen-
trated luminal fluid subjected to ultracentrifuga-
tion at a solution density of 1.063 g/ml. These
results demonstrate that the distribution of
protein constituents in concentrated uterine
luminal fluid is not the same as in rat plasma.
(Aided by a grant from the Natl. Insts. of Health,
PHS.)

497. Thyroxine-binding capacity of serum in
pregnancy. JacoB Rossins (introduced by
J.E. Raut). Natl. Inst. of Arthritis and Metabolic
Diseases, Natl. Insts. of Health, Bethesda, Md.
Since an increase in serum protein-bound iodine

(PBI) has been noted in normal pregnancy

(HEINEMANN, et al. J. Clin. Invest. 27: 91, 1948),

the possibility of a concomitant change in

thyroxine-binding capacity of the alpha globulin
which binds thyroxine (TBP) was investigated.

Serum was obtained from 4 normal young women

and 3 normal young men, and from 9 women in the

$th month of uncomplicated pregnancies. The
serum thyroxine levels, assuming all PBI was

thyroxine, were .055-.089 ug/ml (mean = .074)

in the normals, and .079-.15 ug/ml (mean = .11)

in pregnancy. I'*!-labeled L-thyroxine was added

so that total serum thyroxine varied from 0.2-5.5
ug/ml. Each mixture was subjected to paper elec-

trophoresis (barbital, pH 8.6, ionic strength 0.1)

by a reverse flow technique. In this method, the

electrophoretic mobility of albumin is balanced by
an opposite fluid flow so that albumin remains at
the point of application. The globulins migrate
toward the cathode and are uncontaminated by
albumin-bound thyroxine. At high thyroxine con-
centrations, the quantity of thyroxine in alpha
globulin reaches a plateau which represents the
thyroxine-binding capacity of TBP. In the normal
sera, TBP could bind from .16 to .24 ug of
thyroxine/ml (mean = .19), there being no differ-
ence between men and women. In pregnancy, the

thyroxine-binding capacity of TBP was .31-.53

ug/ml (mean = .41). The increase in binding

capacity in pregnancy was relatively greater than
the increase in serum PBI.

498. Heart pain: its mechanisms and relief
in relation to the small vessels of the heart
and nerves. JosEPH T. Roperts. VA Hosp.,

eer

AMERICAN PHYSIOLOGICAL SOCIETY

153

and Univ. of Buffalo School of Medicine,

Buffalo, N. Y.

While watching the blood vessels of nerves with
the quartz-rod method of translumination, we
have seen the vasa nervorum in the left arm of
dogs and rabbits constrict after ligation of a
coronary artery. This has not been seen yet in
right arm. This confirms our previous belief that
ischemia of somatic nerves lowers the threshold
for pain or other sensory, motor and autonomic
changes in the area of referred pain. Reflex
ischemia of vasa nervorum near motor and auto-
nomic axones in the mixed nerves of the arm is
believed to help explain the common finding of
Dupuytren’s contracture and other types of
shoulder-hand syndrome after a myocardial in-
farction. This mechanism helps explain the great
variability of heart pain clinically, the occasional
absence of cardiac pain with infarctions, the usual
freedom of heart pain in uncomplicated cardiac
hypertrophy, the common loss of angina after
infarction, and other problems. Such a theory for
referred heart pain helps explain relief of cardiac
pain at times by nitrites and other vasodilating
drugs, by local anesthesia of the painful area, by
psychotherapy including group therapy and by
some surgical attempts to revascularize the
ischemic heart. These include 1) arteriolization of
the coronary sinus, veins and arteries, and 2)
endocardial puncturing to exaggerate the
Thebesian type of anastomotic vessels.

499, Relation between renal oxygen consump-
tion, carbon dioxide production and hydro-
gen ion secretion. KATHLEEN E. RoBERTs,
J. W. PoppELt AND Henry T. RanpaALt (intro-
duced by E. F. DuBois). Memorial Ctr. and
Cornell Univ. College of Medicine, New York
City.

According to present concepts the hydrogen ion
secreted by the renal tubular cells is derived
largely from carbonic acid produced metabolically
by renal tissue. If this concept is correct the
amount of titratable acid and ammonia (as an
index of hydrogen ion secretion by the kidney)
would be limited by the amount of renally pro-
duced carbon dioxide which is available. Since
the amount of carbon dioxide produced is also
dependent on oxygen consumed the hydrogen ion
secretion may, therefore, depend also on renal
oxygen consumption and renal blood flow. The
experiments herein reported were carred out on 17
unilaterally nephrectomized dogs to determine
maximum renal capacity for hydrogen ion secre-
tion and to quantitate the relationship between
hydrogen secretion, carbon dioxide production
and oxygen consumption of the kidney. The results
of these studies are as follows; on animals with
renal blood flows ranging between 200-300 cc/min.,

154

the oxygen consumed averaged 245 uM/min. (range
110-400), the carbon dioxide produced averaged
215 wM/min. (range 105-400) and maximal
hydrogen ion secretion averaged 200 wEq/min.
(range 90-285). In individual experiments in
acidotic, phosphate loaded animals the amount of
excreted ammonia and titratable acids was never
greater than 285 ywEq/min. (one kidney) and
approached, but never exceeded, the amount of
carbon dioxide metabolically produced. Because
of its higher bicarbonate content, renal venous pH
averaged 0.02-0.04 px units higher than arterial
blood. Administration of triiodothyronine did not
consistently affect these values, despite marked
increases in total body oxygen consumption.
These studies suggest that the total secretion of
titratable acid and ammonia, in situations optimal
in other respects, is limited within the bounds of
renally produced carbon dioxide and is thus
dependent also on renal blood flow and oxygen
consumption.

500. Production of skin lesions in swine by
the B” (n, a) Li’ reaction. J. 8. RoBERTsoN,
V. P. Bonp,* E. P. CRonx1ITE anp O. D. East-
ERDAY.* Med. Dept., Brookhaven Natl. Lab.,
Upton, N. Y.

Following neutron capture therapy some pa-
tients developed severe epidermitis in the irradi-
ated areas. In a study of relative roles of various
radiations in causing skin lesions, swine were
exposed under conditions simulating patient treat-
ments. Following a control irradiation with
5 X 10" n/cm? to one side of the head, 6 animals
were injected with 30-60 ug B’/gm of body weight
intravenously and irradiated with 5 X 10!2 n/cm?
on the opposite side of the head. A borax: glucose
solution with a molar ratio of 1:2 was used. Ther-
mal neutron fluxes were measured by activation of
gold foils placed at the proximal skin surface. In
separate experiments boron concentration in
selected ¢tissues of 3 swine were determined at
intervals, using a quinalizarin method. The aver-
age boron concentrations in the skin approximated
the average for the body. 30 ug B'/gm and 5 X 10”
n/cm? gives about 1500 rep from a and Li particles.
The proximal skin surface also received approxi-
mately 600 rep from gamma rays and from the cap-
ture of neutrons by hydrogen and nitrogen. Radia-
tion at the distal skin surface was negligible.
Marked edema developed within 24 hr. on the
postinjection side. At about 10 days a severe
epidermitis developed on the postinjection side,
whereas on the control side there was minimal
erythema and epilation. The eye was severely
affected and in 1 animal there was a necrosis of
the tongue on the postinjection side. These studies
appear to establish the important role of heavy
particles resulting from the B" (n, a) Li’ reaction

FEDERATION PROCEEDINGS

Volume 1§

in producing the skin lesions observed in neutron
capture therapy. (This research was supported by
the U.S. Atomic Energy Commission.)

501. Disturbances unrelated to _ mitotie
poisoning induced by colchicine in mam.
mals. Pau, F. Rosinson AND RIcHARD R,
RunGE (introduced by CHaRLEs G. WILBER),
Chemical Corps Med. Labs., Army Chemical
Ctr., Maryland.

Since the discovery of the mitotic-inhibiting
properties of the plant alkaloid, colchicine, much
research has been directed toward the control and
utilization of this natural substance in mitotic
studies. Little work has been accomplished on
the general pharmacological and _ toxicological
properties of the compound. While experimenters
were conducting research on the mechanism of
mitosis, using colchicine, they observed an im-
portant series of findings that have no apparent
relation to the mitotic arrest caused by the plant
alkaloid. In the present work, various animals
were injected with colchicine by different routes,
Death occurred within several hours to several
days. Vomiting, diarrhea, bloody stools and the
exudation of large amounts of porphyrin around
the eyes and nose were the most evident signs of
toxicity. The Lpd’s for various species follow:
rats, 2.2 mg/kg; cats, 1.0 mg/kg; goats, 1.0 mg/kg.
No changes in the following blood constituents in
dogs or goats were found during colchicine poison-
ing: cholesterol, calcium, or nonprotein nitrogen.
Of particular interest are the changes in glucose
metabolism in goats and dogs during poisoning.
Glucose tolerance tests were done in various
animals before and after injection with sublethal
doses of colchicine. These tests showed some very
significant changes in the level of glucose metabo-
lism. In both the dog and goat there was an in-
crease 1 hr. after poisoning in the tolerance of
glucose. Twenty-four hours after poisoning,
another tolerance test was done on the same
animals which then showed a diabetic-type glucose
curve. Pathological examination showed evidence
which leads us to suggest that, shortly after
injection of colchicine, there is a stimulation of
pancreatic action followed later by destruction of
the insulin-producing glands.

502. Positive orientation to light by the star-
fish, Asterias forbesi. Morris RockstEIN.
Dept. of Physiology, New York Univ. College of
Medicine, New York City and Marine Biological
Lab., Woods Hole, Mass.

Twenty intact and 20 completely ‘eyeless’ star-
fish were placed in a dark room in the darkened
portion of a tank of running sea water, } of which
was illuminated by a shaft of white light (100 W
Mazda at 18 in. from the tank) and allowed to dis-

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March 1956

tribute themiselves at random. In most of 7 trials,
as many or more of the ‘eyeless’ as normal animals
were found in the lighted portion of the tank.
Similar results occurred with 26 of each kind of
starfish exposed similarly to light from a (60 W
red safety lamp. Extracts into 2% digitonin were
made of pigments from a large number of pigment
bodies, as well as from adjacent dorsal skin, from
starfish previously dark adapted for 18 hr. These
showed a similar absorption spectrum, with a
pronounced maximum at 320 my and a broad
secondary plateau at 460-500 my, and were sensi-
tive to white (Mazda) light. The biochemical data
confirm the fact that the dorsal skin as well as the
terminal pigment bodies of the rays of the starfish
are involved in positive light orientation of this
species to artificial white light.

503. Effect of respiration and previous cycle
length on pulse wave arrival time. SIMon
Ropgarp. Univ. of Buffalo Chronic Disease
Research Inst., Buffalo, N. Y.

Sounds are generated with the arrival of the
pulse wave at an artery compressed by a blood
pressure cuff. These were recorded using the elec-
trocardiographic R wave as a timing signal. The
time of onset of these sounds in the cardiac cycle
fluctuates slightly during normal respiration. In
pulmonary congestion these fluctuations become
marked, with sounds during expiration occurring
as much as 0.5 sec. in advance of a comparable
beat during inspiration. Duration of the preceding
RR interval also has an effect on arrival time, the
effect being notable in atrial fibrillation. After a
long RR interval the sound comes relatively soon
after the QRS; after a short RR interval the
sound is delayed and weak, or absent. After 2
successive short RR intervals the sound appears
earlier than anticipated and it is more intense
and prolonged. These data suggest an effect of
stroke output. In inspiration the pulmonary
capillaries tend to fill, left ventricular filling is
reduced and a weak arterial pulse wave with
reduced velocity is generated. During expiration
pulmonary capillary blood forced into the left
atrium enhances ventricular filling, creating a
greater stroke output and a strong, rapid pulse
wave. During a short RR interval left atrial
emptying is incomplete, left ventricular filling is
poor and a weak slow wave is generated; after a
second short RR interval the congested hyper-
tensive left atrium injects an increased volume of
blood into the left ventricle and the enhanced
stroke output generates a stronger, faster wave.
The celerity of ventricular contraction probably
also plays a role. The arrival time of the arterial
sound may thus provide significant data concern-
ing pulmonary and atrial congestion and stroke
output.

AMERICAN PHYSIOLOGICAL SOCIETY

155

504. Quantitative clinical measure of cardio-
respiratory dysfunction based on un-
dulations in blood pressure. Simon RopBarp,
JAN CIESIELSKI* AND CHARLES I. Ou1n.* Univ.
of Buffalo Chronic Disease Research Inst.,
Buffalo, N.Y.

The amplitude of the respiratory undulations
in the pulmonary arterial pressure in patients is
correlated with the severity of cardioventilatory
dysfunction, the undulations being more marked
at higher pulmonary arterial pressures (J. Lab. &
Clin. Med. 42:939, 1952). This estimate of cardio-
respiratory impairment is now shown to be de-
termined with facility by indirect measurement of
the systemic arterial pressure. Careful adjustment
of the cuff pressure at slightly above systolic per-
mits selection of a level at which only an
occasional Korotkoff sound is heard; this occurs
during expiration. As the cuff pressure is lowered
by increments of 1 or 2 mm Hg (using a water
manometer) the incidence of arterial sounds
increases gradually until finally a point is reached
at which every heart beat is represented by an
arterial sound. The difference in pressure between
these 2 points is the value of the respiratory
oscillation in a given subject. A similar procedure
determines the diastolic undulation; at the lowest
cuff values arterial sounds are heard only during
inspiration. In normal subjects the respiratory
undulations average 2-3 mm Hg. Patients with
primary pulmonary disease (bronchiectasis,
emphysema, chronic cor pulmonale) show fluctua-
tions up to 25 mm Hg, depending on the degree
of airway dysfunction. In heart disease (hyper-
tensive, rheumatic, arteriosclerotic), fluctuations
range 6-20 mm Hg, being higher with decompensa-
tion and presumed pulmonary capillary conges-
tion. The undulations probably represent
simultaneous variations in intrathoracic pressure.
With expiration, the rising intrathoracic pressure
is transmitted to the aorta and its branches; with
inspiration, intrathoracic and aortic pressures fall.
The degree of cardiorespiratory embarrassment
is apparently associated with ventilatory effort or
dyspnea; the ventilatory undulations therefore
provide a quantitative estimate of dysfunction.

505. Paper electrophoresis of animal hemo-
globins. Grratp P. RopNAN AND FRANKLIN
G. Esavau, JR. (introduced by Jack D. Myrrs).
Natl. Insts. of Health, Bethesda, Md.

When hemoglobin prepared from a variety of
animal sources was subjected to paper electro-
phoresis, differences were found both in the
number of components electrophoretically sepa-
rable from individual animal hemoglobins and in
the mobility of these components relative to
human A, S and C hemoglobin. Under the condi-
tions employed (spinco electrophoresis cell run

156

at room temperature, with veronal buffer of pu
9.2 and 0.03 ionic strength, voltage constant at
270 v. and average current of 0.5 ma across each
strip) save for the horse and 1 of 5 sheep, which
possessed 2 components, all the mammalian
hemoglobins studied were found to have single
components, some with anodic mobility similar
to that of human A hemoglobin (chimpanzee,
rhesus monkey, pig, sheep, goat, cat, mouse, rat,
hamster), some with greater mobility (macaque,
horse, guinea pig), and some with lesser mobility
(llama, cow, dog, rabbit). Chicken and duck
hemoglobins were each found to have 3 electro-
phoretic components, robin hemoglobin 2 com-
ponents, and penguin and pigeon hemoglobin
single components. Hemoglobins prepared from
each of 3 different species of turtles possessed 2
widely separated bands, while that of a 4th species
was found to have 3 components. The single hemo-
globin components of the black snake and water
snake exhibited but slight cathodic mobility, those
of the toad and frog marked anodic mobility, the
former equal to that of human A hemoglobin, the
latter greater. The electrophoresis patterns ob-
tained were reproducible in the same animal and
in different individuals of the same species.

506. Cardiovascular response to prolonged,
repetitive stimulation of the stellate gan-
glion in the dog. Wayne G. Rouse* anp
Water C. Ranpauu. Dept. of Physiology,
Stritch School of Medicine, Loyola Univ.,
Chicago, Til.

In. a report before the Society last year we
stressed the primary importance of the sym-
pathetic innervation of the heart in controlling
the force of myocardial contraction. This work
has been extended by prolonging the period of
repetitive electrical stimulation of the stellate
ganglion in open chest dogs to determine the
character of the cardiovascular response and to
learn hosv long the response could be sustained.
The importance of frequency of stimulation soon
became apparent. A unipolar electrode was affixed
to the caudal pole of the stellate ganglion and
square wave pulses of 2 to 80/sec. in frequency,
10 msec. duration, and from 1.5 to 6.0 v. (moni-
tored by cathode ray oscilloscope) were delivered.
Blood pressure was recorded using a Sanborn
electromanometer and hot stylus recording sys-
tem. The sympathetic trunk was cut below the
T-2 level to eliminate splanchnic constriction and
adrenal secretion. Stimulation at a frequency of
20/sec. or greater resulted in a prompt increase
in systolic pressure, accompanied by lesser or no
increase in diastolic pressure. Frequently no
change in heart rate occurred. During prolonged
stimulation systolic pressure tended to return to
control levels within a relatively few minutes even

FEDERATION PROCEEDINGS

Volume i

though stimulation continued. With frequencies
of 2/sec., while other parameters remained the
same, the onset of blood pressure rise was slower
but reached approximately the same maximum
which was maintained for periods of several hours,

507. Adrenocortical inhibition by Amphenone
‘B.’ Greorce ROSENFELD AND WILLARD PD,
Bascom (introduced by E. HarpENBERGR),
Naval Med. Research Inst., Bethesda, Md.

The acute and direct influence of Amphenone on
steroidogenesis and on several specific enzymatic
systems involved was investigated by concurrent
perfusion in vitro of groups of 4 exposed calf
adrenals and 4 contralateral control glands with
a well-oxygenated artificial medium containing
either a) ACTH or b) steroid substrates. The
extracted effluent steroids were fractionated by
paper partition chromatography and the eluates
assayed by the Porter-Silber (Compounds F and
E), blue tetrazolium (Compound B), and Zimmer-
mann (Cj9-ketosteroids) reactions. The effect of
Amphenone and other inhibitors on glandular
metabolic activity was explored by anaerobic
(Nz) perfusion with the artificial medium con-
taining 2,3,5-triphenyltetrazolium and measure-
ment of the foramzan deposited in the exposed and
control adrenals. A _ striking suppression of
steroidogenesis by Amphenone (7.5 X 10—‘ M) was
evidenced not only by the appreciable diminution
in the biosynthesis (and not the release) of Com-
pounds F, E and B (73%-90%) but also by the
decisive reduction in the ability of the exposed
glands to carry out 118-, 17a-, and 21-hydroxyla-
tions as well as the oxidation of the A5-36-hydroxyl
group to the A‘-3-ketone (65%-90%). The inhibi-
tion of the latter reactions not requiring the
mediation of ACTH and the suppression of the
‘basal’ corticoid production both indicate that
Amphenone exerts its depressant action on the
functional capacity of the cortical cells per se.
In marked contrast to the classical inhibitors ex-
amined, Amphenone induced a substantially larger
decrement in the biosynthetic processes than in
the metabolic ones, and appears to be a relatively
selective inhibitor of the steroidogenic mechanism.

508. ‘Toxic factors’ in burns. Sot Roy RosEn-
THAL, FRANK J. FINAMORE,* F. R. HUNTER AND
AuBErT D. Wiurams.* Inst. for Tuberculosis
Research, Univ. of Illinois and the Dept. of Pre-
ventive Medicine, Univ. of Illinois College of
Medicine, Chicago.

A method for the collection of ‘pocket fluid’
from burned animals has been reported (Federation
Proc. 14: 77, 124, 1955). These experiments were
extended and an 80% ethanol insoluble fraction
from the dialyzable portion of the crude ‘pocket
fluid’ was obtained. Tests with this precipitate

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LOSEN-
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pocket
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March 1956

from ‘pocket fluid’ of burned rats showed a lethal
effect. Intracranial injection of 51 mice resulted
in 44 deaths. Studies with intracranial injections
of a comparable precipitate from ‘pocket fluid’
of control (unburned) rats resulted in the death
of 2 of 9 mice. Control intracranial injections of
water or saline resulted in the death of 2 of 27
mice. Neither the quantity of histamine, Nat,
K* nor Ca** in the 80% ethanol precipitate is
responsible for the observed lethal effects. Beck-
man spectrophotometric analysis of solutions of
the 80% ethanol precipitate reveals the presence
of materials with absorption maxima in the region
of 260 my. Under the proper conditions, ultra-
violet absorbing material can be separated by ion
exchange column chromatography and toxicity
studies of these materials is in progress. In pre-
liminary chronic experiments, the crude ‘pocket
fluid’ from burned rats was injected into four 150-
200 gm rats for periods of 2-3 wk. This treatment
resulted in the death of all rats tested. (Aided in
part by a contract between the Office of Naval
Research, Dept. of the Navy and the Univ. of
Illinois (NR 114 161).)

509. Production of hydrogen peroxide during
the reactions between oxyhemoglobin and
certain reducing substances. H. H.
RosTtorRFER AND M. J. Cormier.* Physiology
Dept., Indiana Univ., Bloomington, Ind., and
Biological Div., Oak Ridge Natl. Lab., Oak Ridge,
Tenn.

A quantum counter, essentially a photomulti-
plying tube cooled with liquid nitrogen, was used
to demonstrate the production of hydrogen
peroxide during the reactions between oxyhemo-
globin and phenzlhydrazine, phenylhydroxyl-
amine or hydroxylamine in the presence of luminol
at pH 8. In the reactions between oxyhemoglobin
and either of the 2 phenyl compounds at the same
concentrations an equally marked liberation of
light occurred. However with a similar concentra-
tion of hydroxylamine light only 1% as intense was
produced. No measurable light was evident in the
comparatively slow reaction between hydrazine
and oxyhemoglobin. Methemoglobin accumulated
when oxyhemoglobin reacted with certain concen-
trations of each of the 4 compounds. A green
pigment and a green precipitate resulted from the
reaction of phenylhydrazine, hydroxylamine and
hydrazine with oxyhemoglobin but no such pig-
ment was produced during the reaction between
oxyhemoglobin and phenylhydroxylamine; instead
nitrosobenzenehemoglobin was formed. The
formation of this compound probably stabilizes
the hemoglobin molecule in some manner which
protects it from the oxidative action of hydrogen
peroxide. It is suggested that methemoglobin and
green pigment are the result of the reaction be-

AMERICAN PHYSIOLOGICAL SOCIETY

157

tween hydrogen peroxide and hemoglobin and that
the velocities of the initial reactions between
the reducing substances and oxyhemoglobin de-
termine the hydrogen peroxide concentration
attained during the reactions. The velocity of the
reduction of methemoglobin by any residuum of
reducing substance also partially determines the
amount of methemoglobin which remains when
the reaction is complete.

510. EEG arousal produced by significant
tones. VERNON Row.Lanp (introduced by
Donatp B. Linpstxy). Schools of Medicine,
Univ. of California at Los Angeles, and Western
Reserve Univ., Cleveland, Ohio.

Cats with cortical and subcortical implanted
electrodes were conditioned to a tone followed by
electric shock to the skin. This tone provoked
EEG arousal when synchronized background
activity was present in relation to spontaneous
sleep, though only rarely did gross movements of
the sleeping animal result. There was no evidence
of adaptation or extinction of this response on
repeated trials. Different tones, even though much
louder than the conditioned ones, provoked similar
EEG alteration when first presented, but after
3-5. trials were ineffective. Behavioral changes in
the waking state showed discrimination of the
same conditioned and neutral stimuli. These
findings confirm other observations that auditory
discrimination is possible during sleep and show
that significant messages are more effective in
producing EEG arousal than non-significant ones.

511. Refractometric determination of total
solids and water content of body fluids.
Miuton E. Rusini* anp A. V. Wo tr. Dept. of
Cardiorespiratory Diseases, Walter Reed Army
Inst. of Research, Army Med. Ctr., Washing-
ton, D. C.

The known correlation between refractive index
and % total solids of plasma provides the basis
for an accurate, rapid and practical determination
of total solids and/or water content of body fluids.
With a dipping refractometer, the instrumental
reading of an unknown (plasma, serum, urine)
minus the reading of distilled water at the same
temperature is called R. Gravimetrically deter-
mined total solids are 8, (gm solid/100 gm water)
and §, (gm solid/100 gm solution). In tests of 118
human and 68 dog serums and plasmas, with Sy
ranging from 6.50 to 12.37, it was found that
R/4.453 = Sy. The coefficient of correlation be-
tween the variables was 0.990; the S.E. of estimate,
0.15 gm % total solids. Rat plasma also fits this
regression. The conversion 8, = 100 S,/(100 + Sw)
is easily carried out separately or by combining
the two equations; or, separate regression equa-
tions directly relating R and §, yield but slightly

158

greater error. Only 1-2 drops of unknown and 3-5
min. are required per determination. An instru-
mental accuracy of +0.02 gm % total solids is
easily obtained with the dipping refractometer.
A small, temperature-compensated hand refrac-
tometer is still simpler and faster to use. Its preci-
sion is about the same as that of the dipping
refractometer; its accuracy is +0.1 gm %
total solids. Refractometric coefficients for urine
are being evaluated.

512. Glucose-6-phosphate and _ 6-phospho-
gluconate dehydrogenase activities of
secondary sex tissues of castrated rats.
GuILrorp G. Rupowpa (introduced by GEORGE
R. MENEELY). Radioisotope Service, VA Hosp.,
Nashville, Tenn.

In order to determine the effect of castration
on the activities of glucose-6-phosphate (G6P)
and 6-phosphogluconate (6PG) dehydrogenases,
assays have been done on the seminal vesicles and
prostates from castrated rats and castrated rats
injected with testosterone propionate. At 24, 48,
72, and 96 hr. after castration the activities of
the enzymes in these tissues were not different
from those of normal controls. The mean activity
of G6P dehydrogenase was 80 u/gm of seminal
vesicle and 60 u/gm of prostate, while the mean
activity of 6PG dehydrogenase was 120 and 70 u
respectively. These tissues showed the same
enzyme activities 3 weeks after castration. Daily
subcutaneous injections of 1 mg of testosterone
propionate to rats 3 weeks after castration resulted
in increased activities of both enzymes. After 3
and 4 injections respectively, the seminal vesicles
increased from 80 to 150 and 190 vu of G6P dehydro-
genase while the prostate increased from 60 to
170 and 195 v. Similarly, the activities of 6PG
dehydrogenase increased from 120 to 190 and
240 u for the seminal vesicles and from 70 to 170
and 200 vu for the prostates. Castration alone did
not change the enzyme activities, while the ad-
ministration of testosterone caused increased
activities.

513. Neural and humoral effects on ventricu-
lar performance. RoBERT F. RUSHMER, JORGE
ANZOLA* AND THEODORE C. WEst.* Depts. of
Physiology and Biophysics, and Pharmacology,
Univ. of Washington, Seattle.

The influences of autonomic nerves and cir-
culating neurohormones on cardiac function was
studied by direct recordings of ventricular dimen-
sions and pressures. Infusion of epinephrine and
levarterenol produced responses which were
quantitatively and qualitatively different from
those induced by direct stimulation of sympa-
thetic fibers to the heart. In general, stimulation
of cardiac nerves produced acceleration of the
heart, reduction in systolic and diastolic dimen-

FEDERATION PROCEEDINGS

Volume 16

sions, reduction in diastolic ventricular pressure
and elevation of systolic ventricular pressure. In
contrast, infusions of both l-epinephrine and
levarterenol (0.4 y/kg/min.) generally produced
bradycardia, increased diastolic and _ systolic
ventricular dimensions and increased diastolic
ventricular pressure. Except for changes in sys-
tolic pressure, the effects of sympathetic and
parasympathetic stimulation on the heart were
the reverse of the changes induced by intravenous
infusion of the corresponding hormones. The
direct effects of the circulating hormones on heart
rate are completely obscured by neural reflexes
associated with changes in systemic arterial
pressure. Therefore, neural control of cardiac
function cannot be simulated by intravenous
infusions of neurohormones. Cardiac responses
during spontaneous cardiac activity (e.g. feeding,
startle, exercise) are more accurately reproduced
by direct sympathetic stimulation than by injec-
tion or infusion of catecholamines. (Supported in
part by a research grant H-716 from the Natl.
Heart Inst., Nat. Inst. of Health, PHS and a
grant from the American Heart Assoc.)

514. Neural pathways of conditioned reflexes
to cortical stimulation. LEester T. Rvt-
LEDGE, JR.* AND Ropert W. Dory. Dept. of
Physiology, Univ. of Utah, Salt Lake City.
Local stimulation of cat cortex for 2 sec. with

0.5-1.0 mA. 2 msec. pulses, 50/sec., served as C8

for forelimb flexion, the CR, to a criterion of

15/25. Is elicitation of CRs by such stimulation

dependent upon intracortical, subcortical or

transcallosal connections; and does cortical condi-
tioning induce a change in other brain regions to
render their subsequent stimulation effective for

CRs, and, if so, is this dependent on maintained

relations with the initially stimulated neurons?

Prior training to criterion with marginal gyrus

stimulation had no (4 Ss) or only slight (2 8s)

effect on trials required for criterion to contra-
lateral middle suprasylvian CS, and no effect on
contralateral posterior ectosylvian conditioning

(3 Ss). Initial training to marginal gyrus CS

greatly reduced trials to criterion from stimula-

tion of the contralateral homologous point (4 of 5

Ss). The latter is concordant with interhemispheric

transfer of visual learning (Myers, R., J. Comp.

Physiol. Psychol. 48: 470, 1955). The conditioned

responsiveness of the homologous marginal gyrus

persisted even though the initially stimulated area
was extirpated prior to the first test (3 Ss). Under-
cutting the stimulated zone, or severing intra-
cortical connections by circumcision of the zone
to a depth of 5 mm caused loss of CRs (one §).

However, in both cases retraining to criterion

was successful, demonstrating the effectiveness

of alternative conduction paths either through the

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March 1956

cortical neuropil, or via subcortical or transcal-
losal fibers. The question of initial postoperative
loss of CRs must await further experimentation.

515. Re-evaluation of potency and properties
of acetyl strophanthidin. A. H. Ryan, W.
WeiL* AnD J. Sprnzia.* Dept. of Physiology and
Pharmacology, the Chicago Med. School,
Chicago, Ill.

Acetyl strophanthidin (AcS) was found to be
cleared exponentially. The mean (geometric)
half clearance time for 16 cats was 28.2+3.27 min.;
extremes 14 and 63 min. In the usual cat assay re-
quiring 30-60 min. sufficient drug is lost during the
assay to introduce considerable error in previous
estimates of potency. To correct this error a modi-
fied assay was devised. The cat under light ether
anesthesia, is given divided doses of AcS until
extrasystoles occur. A waiting period of 30 min.
follows, to permit some clearance of drug. AcS is
again given until extrasystoles and the amount
recorded. Divided doses are continued until the
heart stops. From the first 2 determinations, half
clearance time is estimated. Using this value, a
computation is made of the net amount of drug
remaining at the moment of death. The mean
(geometric) lethal dose of crystalline AcS (kindly
supplied by Eli Lilly) was 0.1376+8.E. 0.0076
mg/kg for 10 cats. The mean dose required to pro-
duce extrasystoles was 53.8%-+8.E. 2.14 of the
lethal dose. A positive correlation (r = 0.67,
P = <.05) was found, between the half clearance
time and the lethal dose. A positive correlation
(r = 0.74, P = .02) was found between the half
clearance time for digitoxin and the dose of AcS
which produced extrasystoles. In light of the modi-
fied assay the high incidence of toxicity in a clinical
trial of AcS reported by Enselberg, Altcheck and
Hellman (Am. Heart J. 40: 919, 1950) may be
attributed, in part at least, to excess doses of the
drug.

516. Effect of insulin on tissue distribution of
intravenously injected L-arabinose. JAcoB
Sacks AND STANLEY Baxsuy.* Dept. of Chem-
istry, Univ. of Arkansas, Fayetteville.

The postulate has been made by Levine et al.
(Am. J. Physiol. 165: 70, 1950; 173: 207, 1953) that
the physiological action of insulin is to facilitate
the transfer of free glucose from the extracellular
phase into the interior of the cells of certain organs
and tissues. They also postulate that insulin has a
similar effect on sugars possessing the same steric
configuration as D-glucose about carbons 1, 2 and
3. Experiments have been carried out in which the
concentrations of pentose and chloride in certain
tissues and organs have been compared with the
plasma concentrations 3 hr. after the intravenous

injection of L-arabinose in nephrectomized cats,

and in similarly treated animals injected with

AMERICAN PHYSIOLOGICAL SOCIETY

159

insulin. The cats were fasted for 24 hr. in order to
reduce the rate of secretion of endogenous insulin
to a low level. In some organs and tissues of the
control animals, the tissue-to-plasma ratios of
pentose and chloride were consonant with distribu-
tion of the injected pentose in the extracellular
phase. In other organs, the pentose concentration
approached that which was consonant with dis-
tribution into the total water content. The injec-
tion of 5 u of insulin/kg b. wt. increased the ap-
parent volume of distribution of the arabinose,
and also the concentration in some of the organs
and tissues which showed an apparent extracel-
lular distribution in the control animals. However,
in those structures in which an insulin effect was
obtained, the final pentose concentration was not
necessarily equal to that corresponding to dis-
tribution in total tissue water. (Supported in part
by a grant from the Natl. Insts. of Health.)

517. Specificity of phosphate acceptor system
during respiration of insect flight muscle
mitochondria. BERTRAM SACKTOR AND DONALD
G. Cocuran (introduced by Leigh CHADWICK).
Med. Labs., Army Chemical Cir., Md.

Flight muscle mitochondria (sarcosomes) of flies
have P/O values approaching 2 when alpha-keto-
glutarate is the substrate. This indicates the
phosphorylation of 1 micromole of inorganic phos-
phate coupled with the electron transport to 1
microatom of oxygen, as is in fact obtained during
the oxidation of alpha-glycerophosphate. With
this latter substrate the optimal concentrations of
the 3 adenosine-5-phosphates were determined.
Twenty-four additional purine and pyrimidine
nucleosides and nucleotides were evaluated as
phosphate acceptors. These included: inosine-5-
mono-, and di-, triphosphates; guanosine-5-mono
phosphate; adenosine-3-monophosphate; uridine-
5-monophosphate; uridine-(2,3)-monophosphate;
uridine-(2,3)-5-diphosphate; thymidine diphos-
phate; thymidylic and cytidylic acids; adenosine;
inosine; guanosine; xanthosine; uridine; cytidine
and thymidine. The desoxy forms of adenosine,
guanosine and cytidine, their corresponding mono-
phosphates, and the nucleic acids, RNA and DNA,
were also assayed. When tested by themselves,
these candidate phosphate acceptors detectably
esterified no inorganic phosphate. Thus the low
P/O value of fly muscle preparations, which is ob-
tained with adenosine-5-phosphates as phosphate
acceptors, appears to be characteristic of this
system rather than due to the requirement of a
different phosphate acceptor. On the other hand,
when the above mentioned candidate compounds
were used in combination with limiting quantities
of adenine-5-nucleotides, several inosine and
uridine nucleotides caused a slight increase in
phosphorylation although this never resulted in a

160 FEDERATION PROCEEDINGS

P/O value greater than 1. This suggests not only
that inosine and uridine nucleotides can be phos-
phorylated but that the sarcosomes possess nucle-
otide transphosphorylases capable of competing
with dephosphorylating enzymes also present in
the sarcosomes.

518. Sources of placental steroids. HILTON
SALHANICK, JOHN Evan JONES AND Davip
BERLINER (introduced by WILLARD ALLEN).
Dept. of Obstetrics and Gynecology, Univ. of
Utah, Salt Lake City.

The steroids which we have isolated from placen-
tal extracts may be divided into three functional
categories: 1) androgen (C;,O2): 4-androsten 3, 20-
dione; 2) Gestagens (C202) : progesterone; 4-preg-
nen- 20a-01/3-one; 3) Corticoids (C2045): cor-
tisone; cortisol; aldosterone; 11-dehydrocorti-
costerone; tetrahydrocortisone; 4-pregnen-17a,
208 , 21-triol,3,11-dione. Evidence based upon sub-
stances isolated from fetal blood extracts will be
presented to support the concept of a fetal source
of androstenedione. The gestagens are assumed to
be formed by the placenta. It is suggested that the
corticoid steroids are concentrated in the placenta
from maternal extra-placental sources. Analysis of
the blood content of 9 placentas ascertained that
at least 10% of the placental weight is accounted
for by plasma. Since the plasma level of corticoids
at parturition is high, it is calculated that at
least $ of the corticoids isolated from our placental
extracts is attributable to the plasma content. It
is further demonstrated that there is a mechanism
for the concentration of cortisone in the placenta.
Fifteen minutes after the administration of
C,4-cortisone, the relative activity of the placenta
to blood was 2.2. This localization may represent
the isotopic dilution of a pool ora specific concen-
trating mechanism.

519. Diabetogenic effects of glucagon. JAMES
M. SapTer,* I. W. F. Davipson* AND CHARLES
H. Best. Banting and Best Dept. of Med. Re-
search, Univ. of Toronto, Toronto, Canada.

The transient action of the hyperglycemic
glycogenolytic factor and the failure to produce
persistent effects are well known. A profound
diabetogenic action has not been reported. An
intense diabetogenic effect is produced, however,
when glucagon, in corn oil, is administered subcu-
taneously to force-fed rats. Ten male rats, 150-160
gm, were force-fed a high carbohydrate diet. By
the 10th day each animal was receiving 18 gm of
solid diet daily. Five served as controls and were
injected with corn oil. Five each received, subcu-
taneously at 6-hour intervals, a total of 1.2 mg of
glucagon (Lilly) daily suspended in corn oil in a
concentration of 3 mg/ml. The blood sugar levels
of the controls remained within normal limits,
with no glucosuria, and an average gain of 28 gm

Volume 1§

during the 7 days. All glucagon-treated animals
developed intense glucosuria within 10 hr. of the
first treatment. The average blood sugars varied
from 350-450 mg% throughout the day. Glucosurig
averaged 4 gm daily while urinary nitrogen
doubled. All these animals lost weight. Three
became ill and were killed before the 5th day,
Pathological changes in the pancreas were exten-
sive. In 2 normal dogs, studied with James Camp-
bell, glucagon in oil produced hyperglycemia
and glucosuria. The probable explanation of thege
findings is that they are due to the slow absorption
of large amounts of glucagon, but there might be
a ‘diabetogen’ distinct from HGF in glucagon
preparations. These findings indicate that many
studies in which glucagon has been ineffective
should be repeated.

520. Effect of weight reduction on physiologic
responses. JOHN SALZANO* AND W. W. Turttig,
Dept. of Physiology, State Univ. of Iowa, Iowa
City.

The effects of weight loss, calculated to occur at
the rate of 2 lb/wk. were studied on 16 subjects
ranging in age from 22 to 48 yr. and varying from
14 to 85% above average weight. Total diets were
prescribed for the subjects who ate all their meals
in the university dining room, under the super-
vision of the dietitian in charge. The experimental
design was as follows: 1) an orientation period of
5 wk.; 2) a 4-wk. period during which present
weight status was maintained; 3) a weight loss
period varying in length with the magnitude of
overweight, followed by 4) a period of 4 wk. during
which the final weight was maintained. The data
showed that there was: 1) a significant decrease in
the total oxygen required to perform a specified
amount of work, a significant decrease in oxygen
debt resulting from the work and a significant de-
crease in resting oxygen. However, when the caleu-
lations are based on per kilogram of body weight,
in most cases there were no such changes in the
above items, 2) a significant decrease both in
diastolic and systolic blood pressures accompany-
ing weight loss in all subjects; 3) a significant de-
crease in reaction time, and 4) in the majority of
subjects no change either in grip strength or grip
strength endurance. (Supported by a grant from
the Cereal Inst., Inc., Chicago, II.)

521. Orthostatic venoconstriction studied by
a miniature balloon technique. Epwin W.
SALZMAN AND SipNEy D. LEVERETT, JR. (intro-
duced by H. O. Parrack). Aero Med. Lab.,
Wright Air Development Ctr., Wright-Patterson
Air Force Base, Ohio.

A study of peripheral venous tone has been car-
ried out in dogs on the tilt table and on the cen-
trifuge using a modification of the method of
Connolly and Wood (J. Appl. Physiol. 7: 239, 1954).

are tae

ume 1§

nimals
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March 1956

Miniaturé*elastic balloons were introduced into
superficial peripheral veins of animals anes-
thetized with a-chloralose and were inflated till
turgid. Pressure in the balloons was recorded with
a transducer having low volume displacement.
Venous pressure was simultaneously recorded
from an adjacent vein. The validity of the system
for demonstrating changes in venous tone inde-
pendent of changes in local venous pressure was
established by noting the response to intravenous
and locally applied sympathomimetic drugs and to
direct stimulation of the lumbar sympathetic
chain. A theoretical analysis of the technique will
be presented. Peripheral venoconstriction during
orthostasis on the tilt table and centrifugation in
the positive-g position has been demonstrated.
This phenomenon is blocked by Dibenzyline
(Smith, Kline, and French), a potent adrenolytic
agent.

522. Anoxia in relation to refrigeration, preg-
nancy, and reticuloendothelial system.
Sretios C. Samaras, Orro J. KLINGER* AND B.
Cart Russum.* Dept. of Pathology, Creighton
Univ. School of Medicine, Omaha, Nebr.

Effects of refrigeration, pregnancy and reticulo-
endothelial blockade upon survival times were
studied in 100 albino or gray-black mice rendered
anoxic by enclosure in sealed 665 ml jars. Experi-
ments extended over 9 months, with controls for
each group. Survival time of mice asphyxiated at
room temperature averaged about 30 min. and
were influenced by the seasons, time of day, age
and specific room temperature. Refrigeration at
7° to 10°C prolonged survival of anoxic animals to
an average of 2 hr., 4 times that of controls. In
spite of known special chemical demands during
pregnancy the average survival time of gravid
mice was the same or slightly longer than controls.
Similar results were obtained in mice asphyxiated
after reticuloendothelial system blockade by
Trypan blue. In hematoxylin-eosin stained slides
of tissues of all experimental and control animals
only parenchymatous degeneration of the liver,
hyperemia and hemorrhage of the lungs with over-
distention, and hyperemia of the spleen and me-
ninges could be correlated with the anoxia. Par-
enchymatous degeneration of the liver occurred
in all refrigerated animals, while pulmonary hy-
peremia and hemorrhage were found in some.
With combined anoxia and refrigeration par-
enchymatous degeneration occurred in all mice,
hyperemia in some; pulmonary hyperemia and
hemorrhage occurred in most, hyperemia alone in
some; in several cerebral hyperemia was present.
We hope that further detailed histologic studies of
the brain now under way and additonal experi-
ments under controlled climatic conditions will
help to interpret observed functional differences.
(Aided by Nebraska Heart Assoc. grant.)

AMERICAN PHYSIOLOGICAL SOCIETY 161

523. Effect of temperature upon rate of cere-
bral energy requirement in neonatal rats.
Freperick E. Samson, JR. AND Nancy DanL
(introduced by J. O. HutcuEns). Dept. of Physi-
ology, Univ. of Kansas, Lawrence.

Neonatal rats poisoned with iodoacetic acid
(0.18 mg/gm) to inhibit glycolysis were subjected
to anoxia for a determined period of time and then
returned to air. They were observed for signs of
viability such as respiratory movements. The
length of time which anoxia could be survived
(survival time) was found to be a function of both
age and body temperature. The younger the ani-
mal, the longer the survival time. Lowering the
body temperature increased the survival time.
This effect of temperature increased with de-
creasing age. With 10-day rats the survival time at
35°C. was 15 sec. and at 10°C. had increased to 60
sec. With 1-day rats the survival time at 35°C. was
20 sec. but at 10°C. had increased to 255 sec. There
is a linear relationship between the logarithm of
the survival time and the body temperature in the
1-5-day-old animals. (The animals which survived
the anoxia succumbed to the iodoacetic acid
poisoning about 30 min.-2 hr. later, depending
upon the body temperature. Death was immedi-
ately followed by intense rigor.) Since the brain is
probably the limiting organ to survival under the
conditions of these experiments and the known
sources of energy to cerebral tissue are abolished,
it is suggested that these survival times are a
measure of the amount of cerebral reserve energy
and the energy requirement rate for cerebral
viability.

524. Onset of active state in potentiated mus-
cular contractions. ALEXANDER SANDOW AND
ManFrep Brust. Dept. of Biology, Washington
Square College of Arts and Science, New York
Univ., New York City.

Using the piezoelectric, cathode-ray oscillo-
graphic method (Kaun anp Sanpow. Ann. New
York Acad. Sc. 62: 137, 1955), studies have been
made of peak tension (T) and time derivative of
tension development (with particular interest here
in maximal value, D, of this function) of maxi-
mally, massively stimulated frog sartorii. When,
in a 1/sec. series of twitches at 20°C, treppe is
maximum, both T and D, relative to original
values, are each increased by about 20 to 25%.
Similar parallel increases in T and D occur in post-
tetanic potentiation, whether the tetanus is intro-
duced in the twitch series at peak of treppe or at
fatigue. Thus, in potentiation of T of such
twitches, or in anion-modified ones (see above
reference), there is an associated increase in rate
of tension onset early in contraction. When rested
muscles are activated by a dual-shock stimulus
the 2nd shock, if early enough, increases D in
comparison with its value due to the Ist shock

162

alone. At 2°C the maximal increase in D, about
4%, occurs when the shock interval is about 20
msec. Corresponding values at higher tempera-
tures are: 10°, 15% at 10 msec.; 21°, 60% at 4 msec.;
30°, 60% at 4 msec. Hence, this way of increasing
D is highly temperature sensitive from 2 to 20°.
The results in general indicate that potentiation
of T is due not only to the previously suggested
prolongation of the active state, but also to ac-
celeration in rate of onset (activation) of this
state. (Aided by grants from the Muscular Dys-
trophy Associations of America, Inc., and by con-
tract with the Office of Naval Research.)

525. Effects of nitrate on normal and dys-
trophic rabbit muscle. ALEXANDER SANDOW
AND Epwarp SrTe1n.* Dept. of Biology, Washing-
ton Square College of Arts and Science, New York
Univ., New York City.

These studies extend to mammalian muscle
previous work which showed that frog muscle
(sartorius) exposed to nitrate-Ringer’s produces
potentiated and prolonged twitches due to a direct
action of nitrate on the muscle fiber membrane,
which in turn causes prolongation of the twitch
active state. Normal rabbit skeletal muscle
(median lumbrical) treated with mammalian ni-
trate-Ringer’s responds to a single maximal shock
(determined beforehand in chloride-Ringer’s) as if
veratrinized; for a) the rested muscle produces a
typical veratrinic mechanical output whose initial
phase is accompanied by asynchronous repetitive
spikes; b) repeated stimulation at 1 shock/sec.
evokes veratrinoid responses of diminishing in-
tensity (but when, thus, pure twitches are ob-
tained they exhibit nitrate effects somewhat as in
frog muscle); and c) rest, after abolition of the
veratrinoid responses, causes restoration of the
veratrinic effects. These veratrinoid properties are
not due to the special features of high temperature
or in cation composition involved in utilization of
the mamnfalian-nitrate Ringer’s. The veratrinoid
effects appear after only a few minutes exposure of
the muscles to the nitrate medium. Eserine, which
curtails effects of veratrine in frog muscle, acts
similarly on nitrate-veratrinoid responses of rab-
bit muscle. Muscles from vitamin E deficient
dystrophic rabbits do not show the nitrate-ver-
atrinoid effect, but their twitches are potentiated,
though not regularly, by nitrate. These results
will be discussed in relation to possible mecha-
nisms in the membrane by which nitrate produces
its various, specific effects on the active state of
muscles of the normal frog and of the normal and
dystrophic rabbit. (Aided by grants from the Mus-
cular Dystrophy Assns. of America, and by con-
tract with the ONR.)

526. Discharge of ACTH from the adenohy-

pophysis of the adrenalectomized rat.

FEDERATION PROCEEDINGS

Volume 16

GrorcE Sayers. Dept. of Physiology, Western

Reserve Univ., Cleveland, Ohio.

Blood ACTH in the adrenalectomized rat ex-
hibits a marked increase during the first few
minutes of ether anesthesia. After 30 min. of ether
anesthesia blood ACTH returns to preanesthetic
levels. Pitressin and epinephrine administered to
the 30-min.-ether adrenalectomized rat induce an
elevation in blood ACTH. Histamine, 3.0% NaCl,
Pitocin, clamping the carotids for 2 min. or sham
stalk section do not increase blood ACTH. A
lyophilized powder of commercial Pitressin
(Parke-Davis * 168941) exhibits the same ACTH
discharging activity per unit of vasopressor ac-
tivity as commercial Pitressin in vials. The ac-
tivity of the powder is stable in 0.01 Nn HCl at 100°C
for 1 hr., but is destroyed in 1.0 n HCl at 100°C
for 1 hr. A purified preparation of vasopressin ob-
tained from Dr. du Vigneaud (AVN-5) exhibited
the same ACTH discharging activity as commer-
cial Pitressin per unit of vasopressin activity.
Extracts of rat neurohypophysis but not spleen,
skeletal muscle or liver induce ACTH discharge.
Each rat neurohypophysis has an ACTH dis-
charging potency equal to about 1 unit of com-
mercial Pitressin. The results are compatible with
but do not prove the thesis that vasopressin
(ADH) is the factor which induces ACTH dis-
charge in adrenalectomized-etherized rats. It re-
mains to be demonstrated that ADH acts directly
on the adenohypophysis. Pitressin is equally
effective in inducing ACTH discharge, whether
given by saphenous vein or by internal carotid
artery.

527. CO, effects on carbonic anhydrase ac-
tivity and electrolytes of hypothalamus and
cortex. K. E. ScHAEFER AND B. Barton. U.S.
Naval Med. Research Lab., U. S. Naval Submarine
Base, New London, Conn.

Carbonic anhydrase activity, K and Na content,
was studied in hypothalamic and cortical areas of
guinea pigs exposed to 15% COs in 21% Os» over
periods up to 7 days. Although both brain tissues
exhibited a small corresponding rise in blood con-
tent, carbonic anhydrase activity of the cortex in-
creased, while that of the hypothalamus decreased
after 7 days of exposure to 15% COs. In the cortex
the sodium level was maintained; the potassium
content, however, declined slightly. In the hy-
pothalamus the sodium content fell significantly,
paralleling the decrease in carbonic anhydrase ac-
tivity in this area. The potassium level was found
only slightly lowered. The potassium sodium
ratios changed in opposite directions, showing ap
increase in the hypothalamic area and a decrease
in the cortex. In the red cell-plasma system the
sodium gradient increased due to a migration of
sodium into the red cells, while the potassium

el;

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ually
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March 1956

gradient decreased correspondingly, involving a
reduction of potassium in the erythrocytes.

528. Ventricular depolarization. ALLEN M.
ScuEeR. Dept. of Physiology and Biophysics, Univ.
of Washington School of Medicine, Seattle.
Multichannel recording techniques permit de-

tailed description of total ventricular activation.
Depolarization begins a few milliseconds before
the onset of QRS at the septal terminations of the
left bundle and advances to the right. Activity on
the right septal surface commences slightly later,
about at the onset of QRS, and moves leftward.
Rapid activation of most of the apical endo-
cardium by the branched Purkinje network leads
to endo-epicardial movement in the free walls.
Epicardial breakthrough is prominent on the
right a quarter of the way through the QRS com-
plex. Half way through QRS, the major portions
of the free wall, except a thin sheet, mostly on the
posterior and lateral left walls, are active. Later
portions of QRS involve spread to the basal left
wall and to the posterior basal septum, the last
area depolarized. Important factors in excitation
are: 1) activation of the endocardium by the
Purkinje fibers conducting at about 1.0 m/sec
(with higher apparent apical velocity due to ex-
tensive branching), 2) mural endo-epicardial move-
ment in the walls at about 0.3 m/sec, and 3) double
envelopment of the septum. Three major electro-
cardiographic vectors result: Ist, from left to right
in the septum; 2nd, base to apex due to mural endo-
epicardial activation and, finally, apex to base in
the upper septum. (Aided by Grant H-1315 from
the Natl. Heart Inst.)

529. Effect of vagal stimulation on crushed
sinus node tissue of the dog’s auricle.
Davin Scuerr, SerRGE BLUMENFELD, EDITH
Rem anp ArtTHUR WEIsz (introduced by I.
S. Kierer). Dept. of Medicine, New York Med.
College, New York City.

Experiments were carried out on 24 dogs in
which the heart was exposed in situ under mor-
phine-Nembutal anesthesia. Stimulation of the
right vagus nerve in the neck with a strong faradic
current caused as usual auricular and ventricular
standstill. In 18 of our 24 experiments after appli-
cation of a surgical clamp over part of the sinus
node area the auricles continued to beat during
vagal stimulaion and the chief effect noted was
A-V block. In 5 of these experiments the rate of
the auricular pacemaker was unchanged before
and during the vagal stimulation. Frequently the
application of the clamp led to the appearance of
a new auricular center; this was seen in 13 of our
24 experiments. In the remaining 5, the form of the
P wave remained unchanged before, during, and
after vagal stimulation. After removal of the
clamp this phenomenon lasted up to 29 min.

AMERICAN PHYSIOLOGICAL SOCIETY 163

These findings appeared although no attempt was
made to clamp off the entire sinus node; however,
they were not present when other parts of the
right or left auricle not containing special tissue
were crushed. There is as yet no explanation for
the lack of response or the poor response of the
auricular pacemaker to vagal stimulation follow-
ing crushing of the specific tissue. (Supported, in
part, by a research grant from the Natl. Heart
Inst., PHS.)

530. Homeostasis during antidiuresis. J. U.
ScHLEGEL AND H. Stone.* Dept. of Surgery,
Div. of Urology, Univ. of Rochester, Rochester,
NY;

Antidiuresis is a normal consequence of trauma
in man. It can be duplicated in its entirety by ad-
ministration of antidiuretic hormone. The conse-
quence of antidiuresis in the face of administration
of water with no solid content is a dilution of the
body fluids with the development of water intoxi-
cation. During antidiuresis the renal excretion of
water is directly proportional to the excretion of
solids. It thus appears reasonable that water re-
tention might be prevented despite antidiuresis,
provided that the intake of solids is sufficiently
high. The present study deals with the effect of
differing concentrations of NaCl and urea, under
a constant water load, on rats that have received
antidiuretic hormone. It was found that a quanti-
tative return of the administered water was ob-
tained if NaCl was present in a concentration of
2-2}%, or urea in a concentration of 4-5%. Salt
retention was induced in addition to water re-
tention by administration of DCA to adrenalec-
tomized rats who in addition received post-
pituitary hormone. Under such circumstances it
was found that homeostasis could not be main-
tained by adding NaCl of any concentration to the
administered water while urea in proper concen-
trations was still capable of preventing water re-
tention. The implications of the experimental
results in regard to antidiuresis of trauma are
discussed and a brief report of the use of urea solu-
tions, as posttraumatic infusions, is referred to.
(Supported in part by contract Nonr-668 Task 7
from the Office of Naval Research.)

531. Comparative aspects of urea excretion in
vertebrates. Bopit Scumipt-NIELsEN. Duke
Univ., Durham, N.C.

Renal excretion of urea in mammals has been
assumed to be entirely passive. The current con-
cept is that urea enters the kidney tubule by
glomerular filtration, that part of the urea diffuses
back through the tubular wall, and that urea is
neither actively secreted nor actively reabsorbed.
However, many studies have brought out evidence
incompatible with this concept. /) In certain
desert rodents (both old and new world species)

164

on high protein diet the urea clearance exceeds
filtration rate. 2) In white rats raised on high
protein diet and drinking 3% urea solution, urea
clearance exceeds filtration rate by 40%. 3) In
white rats and desert rodents urea clearance de-
creases about 50% relative to filtration rate when
they are transferred to low protein diet. 4) In a
grazing camel on medium protein diet, approxi-
mately 40% of the filtered urea is excreted, while on
low protein only 1% is excreted. These variations
are independent of filtration rate, plasma urea
concentration, and state of hydration. 5) In man
urea clearance varies selectively with variations in
dietary protein level (NIELSEN AND Bane. Scand.
J. Clin. Lab. Invest. 1: 295, 1949). These and other
findings suggest that urea excretion can be actively
regulated in mammals. The concept of active regu-
lation in mammals is also reasonable in view of
many various mechanisms found in other verte-
brates. Active reabsorption of urea is present in
Elasmobranches and active secretion in frogs (all
species of Rana so far examined). Phylogenetically
it seems unlikely that mammals should have
entirely discarded efficient regulatory mechanisms
in dealing with their major nitrogenous excretory
product.

Knut
Duke

the camel,
of Zoology,

532. Water storage in
Scumipt-NreLsEn. Dept.
Univ., Durham, N.C.
The camel has exceptional abilities to withstand

water deprivation and desert heat. One explana-

tion frequently offered is that the camel stores
water for use when need arises. This hypothesis
has been supported by the presence of unusual sac-
like structures, erroneously called ‘water sac,’ in
the camel’s stomach (rumen). The fluid found here
is alleged to be stored water and used as an
emergency water supply by desert Arabs. The
amount of fluid that was found in the rumen and
digestive tract of desert camels (Camelus drome-
darius) Was not greater than normally found in
other ruminants such as cows. The rumen sacs
contained semi-solid masticated feed. While the
rumen of other ruminants has no glands, the rumen
sacs of the camel have simple tubular glands.
There was no difference in the chemical compo-
sition of fluid from the sacs and the general rumen
fluid, all samples being isotonic or somewhat hypo-
tonic to blood. The fluid had no conspicuous
similarity to the available drinking water. It is
proposed that the fluid has a digestive role and no
function in water storage. After the camel ingests
large quantities of water, the rumen fluid regains
its usual composition in about 2 days, indicating
complete distribution of the ingested water in the
body. From the drinking pattern as well as studies
of the water compartments (plasma volume, extra-
cellular, and intracellular spaces) there was no

FEDERATION PROCEEDINGS

Volume 1§

indication that camels drink more than necessary
to bring the water content of their bodies up to
normal after periods of water deprivation.

533. Effects of increased cardiac work on
coronary blood flow and left ventricular
metabolism: nicotine. JERRY E. Scuamir-
THENNER,* CECILIA RIEGEL AND JOSEPH H,
HAFKENSCHIEL. Lankenau Hosp., Philadelphia,
Pa.

Dogs anesthetized with morphine pentobarbital
urethane were given infusions of 0.9% sodium
chloride, nicotine base 2-6 w/kg/min and 14-2
u/kg/min. Studies were made of stroke volume,
cardiac rate, mean arterial pressure, coronary

blood flow, arterial and cardiac venous (coronary |

sinus) glucose, lactate, pyruvate and oxygen
levels. The lower dose of nicotine had effects
similar hemodynamically to those produced by
intravenous infusion of epinephrine 0.01-0.30
u/kg/min. Left ventricular carbohydrate extrac-
tion was unchanged. Higher doses cf nicotine in-
creased cardiac rate but not left ventricuiar work.
Coronary blood flow and left ventricular oxygen
consumption increased. Although arterial blood
glucose rose in a few instances, there was no ap-
parent change in carbohydrate extraction. The
data accumulated support the thesis that left
ventricular usage of carbohydrate is determined
by arterial concentration and not by increased
cardiac work. (Supported in part by a research
grant (H1817) from the Natl. Heart Inst., PHS,
and by a grant from the Tobacco Industry Re-
search Committee (QH17).)

534. Effect of pilocarpine on rat salivary gland
amylase content. LEon H. ScHNEYER AND
Cuar.LorTe A. ScHNEYER.* Depts. of Physiology
and Clinical Dentistry, School of Dentistry, and
Med. College of Alabama, Univ. of Alabama,
Birmingham.

Administration of pilocarpine has generally
been observed to result in an increased rate of
salivary secretion accompanied by depletion of
gland stores of specific secretory proteins (e.g.
amylase). In the experiments to be reported here,
the expected increase in secretory rate and de-
pletion of amylase stores were observed for the
parotid gland of Norway rats after subcutaneous
administration of pilocarpine. In the case of the
submaxillary gland, on the other hand, although
pilocarpine administration resulted in the ex-
pected increased secretory rate, a rapid and signifi-
cant increase in the level of amylase in the gland
was observed. This increase could be detected
within 15 min. after initial administration of the
drug. Determination of gland amylase at selected
intervals thereafter showed further increases; by
the end of 33 hr., the amylase content of the pilo-
carpine-stimulated gland was 10 times that of the

col

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essary
up to

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odium

14-20
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xygen
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. The
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earch
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March 1956

control. Stmmulation of submaxillary secretion by
means other than pilocarpine (electrical stimula-
tion or feeding) did not produce an increase in
gland amylase content. (Supported by funds pro-
vided under Contract AF 18(600)-623 with the
USAF School of Aviation Medicine, Randolph
Field, Texas.)

535. Membrane conductance changes in
isolated nodes of Ranvier. Gorpon M.
ScHOEPFLE. Dept. of Physiology, Washington
Univ. School of Medicine, St. Louis, Mo.
Responses of an isolated node of Ranvier were

recorded by means of a ‘double air gap’ technique

modified after Tasaki such that stimulating and
recording circuits are separated to a much greater
extent than is possible with the ‘single air gap’
method previously employed. The magnitude of
the single fiber action potential elicited during or
after polarization has been investigated as a
function of intensity and duration of anodal
polarizing current applied to normal and depressed
fibers. While the time course of change in spike
height cannot be correlated with any change in
membrane potential induced by polarization, the
kinetics suggest that the ‘sodium inactivation’
mechanism of Hodgkin and Huxley accounts for
the results obtained. By way of further support it
is observed that brief direct current pulses elicit
changes of membrane potential during activity
which indicate, qualitatively at least, that in-
crease in spike height during anodal polarization
of depressed nerve is associated with a greater
than normal change in membrane conductance.
Quantitative determinations are enormously com-
plicated by the finding that, near peak of the
action potential, a testing pulse of minimally per-
ceptible effect induces an appreciable change in
membrane potential in a direction opposite to that
expected. This is taken as consistent with the
concept that these these minimal changes in mem-
brane potential are associated with finite changes
in sodium conductance. (Aided by grant B-173,
Natl. Insts. of Health, PHS.)

536. Measurement of ion flow across the ex-
citable and nonexcitable membranes of a
single cell. ERNEST SCHOFFENIELS AND Mario
ALTAMIRANO (introduced by Davin NacHMAN-
soHN). Dept. of Neurology, College of Physicians
and Surgeons, Columbia Univ., New York City.
A new type of preparation of electroplax of

electric eel will be described in which the influx

and outflux of different ions through the innervated
and noninnervated membrane of a single cell are
independently measured. An electroplaque is dis-
sected and mounted in a lucite chamber formed by

2 pools separated by thin sheets of plastic. The

sheets have a small window of the dimensions of

the cell to be studied. The electroplaque is tightly

AMERICAN PHYSIOLOGICAL SOCIETY 165

fixed in the window with its innervated face in
contact with the solution of 1 pool; the nonin-
nervated membrane contacts the solution of the
opposite pool. Any substance transferred from 1
side to the other has to cross the 2 membranes of
the cell. The solutions of both pools are well
mixed by a pump or continuously removed by a
circulating system. Suitable electrodes allow the
recording of the electrical potentials of the cell.
Thus, a preparation has been developed which
permits the study of ion flux across the conducting
membrane of a single cell and the effect of physical
and chemical factors on this process in a direct
way. Two general types of experiments have been
performed so far. In some the rate of transfer of a
radioactive element from 1 pool to the other was
measured. In other experiments the electroplaque
was loaded with radioactive material and the loss
of activity in inactive solution was followed.

537. Biochemical and cytological effects of
ACTH on surviving adrenocortical tissue.
E. ScHONBAUM AND W. G. B. CAssELMAN (intro-
duced by E. A. Setuers). Dept. of Physiology
and the Banting and Best Dept. of Med. Research,
Univ. of Toronto, Toronto, Canada.

ACTH is known to stimulate steroid formation
by adrenal tissue in vitro provided calcium is
present in the incubation medium. Likewise, glu-
cose or one of its metabolites is known to enhance
this effect. In the studies reported here, versene
was found to abolish the effect of ACTH, indicat-
ing that at least calcium must be present in ionic
form. Bicarbonate buffer results in greater stimu-
lation than either phosphate- or tris-buffer. Glu-
cose can be replaced by fructose. Succinate, un-
like glucose, does not enhance the ACTH effect
significantly. Such observations as well as evidence
from the literature suggest that ACTH might
affect the balance between the amounts of glucose
oxidized in the Krebs cycle and the amounts
needed for an increased steroid synthesis. There is
good correlation between the increase in steroid
formation due to ACTH added in vitro to surviving
rat adrenal tissue and the cytological changes in
z. fasciculata and z. reticularis during the incu-
bation period. ACTH causes increased dispersion
and apparent decrease in amount of demonstrable
lipid, not observed in the absence of ACTH. A
partial activation of the adrenocortical tissue was
observed when even brief Nembutal anesthesia
was used but not when the rats were decapitated.
(Supported by a grant, DRB 113, from the De-
fence Research Board of Canada.)

538. Stimulation-length sequence and
length-tension diagram. Brron A. Scuort-
TELIUS (introduced by WattTerR 8. McCLeLuan).
Dept. of Physiology, Univ. of North Carolina,
Chapel Hill.

166

Previously reported (Am. J. Physiol., 179: 491,
1954) active length-tension diagrams of rat gas-
trocnemii exhibited a marked secondary peak of
the descending curve between 100 and 110% resting
length. It appeared likely that the secondary
maximum was either a) an artifact of stimulation
sequence, i.e. physiological fatigue, or 6) the effect
of change, with stretch, in attachment angle of
individual fibers. In order to test hypothesis a,
stress-strain data of gastrocnemius muscles, at rest
and in contraction, were obtained from 3 groups
of adult male rats. Muscles of the first group were
tetanized only once and at a single stimulation
length. Thus the curves obtained were free of
sequence and fatigue artifacts. Muscles of both
other groups were stimulated at each length ex-
amined (88-127% resting length). Gastrocnemii of
the second group were tetanized, at lengths less
than resting, in an ascending length sequence.
Group three muscles were examined by previously
reported techniques. The secondary peak was evi-
dent in active and total length-tension curves of
all groups except the first. Its presence in group
two curves was dependent upon the sequence of
stimulation at resting length. In addition, multi-
ple stimulation, as used in groups two and three, in-
creased the extensibility observable in the passive
tension curves. It is concluded that the secondary
maximum is a stimulation-length sequence arti-
fact, not exclusively attributable to fatigue.
Rather, it appears to derive from a shift in optimal
stimulation length, i.e. equilibrium length.

539. Effect of thyroxin on stimulus frequency-—
tension output relationship in the neuro-
muscular system. NEENA B. ScHWARTZ AND
Ann H. InGoup (introduced by C. I. Reed).
Dept. of Physiology, Univ. of Illinois, College of
Medicine, Chicago.

We have shown (Am. J. Physiol., 182: 5, 1955)
that hypothyroidism increases tension and hyper-
thyroidisfd decreases tension, when the gastroc-
nemius is stimulated at subtetanic frequencies, the
stimulus electrodes being directly inserted in the
muscle. Doubt about whether or not the stimula-
tion was via intramuscular nerves makes it diffi-
cult to draw conclusions about the site of action of
thyroxin. Accordingly, the following experiment
was performed. Muscles of hypothyroid, euthyroid
and hyperthyroid rats were stimulated in situ at
frequencies of 15, 30, 45, 60, 90 and 120/sec. (super-
maximal intensity) and the maximum tension per
gram muscle determined at each frequency. One of
3 stimulation routes was used for each animal:
1) electrodes on sciatic; 2) electrodes in muscle;
3) electrodes in curarized muscle. Results obtained
by routes 1) and 2) were similar, the tension at a
given frequency being low for hyperthyroids and
high for hypothyroids, in comparison with nor-

FEDERATION PROCEEDINGS

Volume 1§

mals. In the curarized muscles, however, the fre-
quency-tension relationship was essentially the
same for all groups. This indicates either that
thyroxin level affects nerve fiber or ending; or that
thyroxin affects muscle fiber or end plate, this
effect being abolished by curare. Measurements at
120/sec. indicate that within a given treatment
group tension fatigue occurs at the same rate re-
gardless of stimulation route, but that fatigue
occurs more quickly in the hyperthyroid rats than
in the others. (Supported in part by a research
grant (B 768-R) from the Natl. Inst. of Neuro-
logical Diseases and Blindness, PHS.)

540. Electrolyte and water diuresis produced
by inactive N*-substituted acetazoleamide
analogs. Wi._uiAM B. Scuwartz, RicHarp
PORTER AND ARNOLD 8. RELMAN (introduced by
E. B. Astwoop). Depts. of Medicine, Tufts Univ,
School of Medicine and Boston Univ. School of
Medicine, Boston, Mass.

It was previously reported that 500 mg/kg doses
of acetazoleamide (Diamox) in dogs produced a
brief profuse diuresis, in which more than ? of the
filtered bicarbonate and more than $ of the filtered
sodium and water were excreted. The effect was
attributed to inhibition of carbonic anhydrase ac-
tivity and the consequent reduction in tubular
secretion of hydrogen in exchange for sodium. In
the present experiments, the N-substituted
methyl and isopropyl analogs of acetazoleamide,
which have little or no carbonic anhydrase in-
hibiting activity, were found to have almost the
same electrolyte effects as the active parent com-
pound. Doses of 10 mg/kg of the analogs had
virtually no effect, whereas a marked bicarbonate
diuresis is always produced by this dose of ace-
tazoleamide. A significant osmotic effect seems un-
likely in view of a) relatively low urine concen-
trations of sulfonamide, and b) failure of equimolar
loads of sodium chloride and sodium bicarbonate
to produce more than a slight diuresis. It is sug-
gested that most of the diuretic action of the large
doses of acetazoleamide, and the entire action of
the inactive analogs, is probably due to the be-
havior of these compounds as strong bases, rather
than to inhibition of carbonic anhydrase. Tubular
reabsorption of these sulfonamides might tempo-
rarily bind enough intracellular hydrogen to re-
duce the amount available for secretion. The
magnitude of the diuresis would then suggest that
most, if not all, sodium is reabsorbed by exchange
with hydrogen.

541. Bile salt absorption in the small in-
testine. G. W. SEARLE AND R. D. Baker (intro-
duced by J. D. Tuomson). Dept. of Physiology,
State Univ. of Iowa, Iowa City.

As a step toward finding out why some bile salts
aid intestinal fat absorption while others do not,

ime 15

he fre-
ly the
r that
or that
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tment
ite re-
atigue
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iced a
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salts
not,

March 1956

information on the absorption of the bile salts
themselves at various levels of the intestine was
sought. Accordingly, 3 acute intestinal loops, each
about 30 cm long, in dogs were used for determin-
ing simultaneously the 30-min. absorption of
cholate. Each loop was filled initially with 25 ml
of 1% aqueous solution of Ox Bile Extract, U.S.P.
(Wilson Labs.), containing about 125 mg of cholate.
The preliminary cannulation of the loops, as well
as the subsequent absorption tests were carried out
with the dogs anesthetized with sodium pento-
barbital. Each loop was rinsed before the test with
warm normal saline. At the end of the absorption
period the loop contents and 3 separate 25 ml
washings were combined and analyzed for cholate
by the method of Irving, Johnson and Kopala.
Net absorption in 30 min. was estimated from the
results in 6 dogs by comparison with the apparent
absorption which occurred when the test solution
was infused and then removed immediately in 2
other dogs. By this method no significant absorp-
tion occurred in 2 adjacent loops, the 1st of which
was begun just caudal to the ampulla of Vater,
whereas a mean of 23% absorption took place in
loops of the terminal ileum. Tests in 2 additional
dogs post-mortem showed a small amount of ab-
sorption in 30 min. which appeared to be of about
the same magnitude in all 3 loops.

542. Macroglobulins in human sera. A. H.
Senon, L. Gyenss, J. Gorpon, M. RicHTER AND
B. Ross (introduced by J. 8S. L. Browne).
Allergy Research Div., McGill Univ. Clinic,
Royal Victoria Hosp., Montreal, Canada.

Three cases of macroglobulinemia have been
investigated. In each case the dilution of the whole
serum with distilled water (1:15) resulted in the
precipitation of a globulin fraction with proper-
ties similar to those reported for ‘macroglobulins’
by Waldenstrom (Acta Med. Scand. 117: 216, 1944).
Ultracentrifugal experiments revealed that the
macroglobulin fractions were heterogeneous. The
sedimentation constants of the various peaks re-
solved in the ultracentrifuge range from 11 to 298.
Free and zone electrophoresis in veronal buffer at
pH 8.6 showed that the macroglobulins were slow
moving gamma globulins in two cases, and in one
case they included minor components identified as
p-and a-2 globulins. Staining with the Schiff rea-
gent revealed that the macroglobulins were rich in
carbohydrate. Preliminary experiments indicate
that a globulin fraction with similar properties is
present in trace amounts in normal serum. Experi-
ments for the further characterization of this ma-
terial by physico-chemical and immunological
methods are in progress. (Supported by a grant
from the Dept. of Natl. Health and Welfare,
Ottawa, and from the Charles E. Frosst Co.,
Montreal.)

AMERICAN PHYSIOLOGICAL SOCIETY 167

543. Splanchnic hemodynamics and O, utili-
zation during hemorrhagic shock. E. E.
SELKURT AND G. A. Brecuer. Dept. of Physi-
ology, School of Medicine, Western Reserve Univ.,
Cleveland, Ohio.

Studies of portal vein and hepatic artery flow
were made in dogs using phasic flow meters during
a standardized hemorrhagic shock procedure.
Analysis of O» content of arterial, portal vein and
hepatic vein blood permitted computation of O»
utilization by the intestine and liver. Portal vein
flow averaged 310 ml/min. and hepatic artery flow
170 ml/min. during control. These decreased to 127
and 100 ml/min., respectively, on hemorrhage to
60 mm, then increased to 155 and 130 ml/min. as
pressure was held at this level for 90 min. On
further hemorrhage to 40 mm portal flow decreased
to 100 ml/min. and hepatic artery flow to 70 ml/
min. On transfusion, portal flow increased to an
average of 470 ml/min. at 30 min. post-transfusion,
despite incomplete recovery of arterial blood pres-
sure. A significant increase in portal venous pres-
sure accompanied this hyperemia. Hepatic artery
flow returned approximately to the control value.
Flows decreased later as pressure fell. Average O2
utilization was: intestine: control, 19 ml/min.; 60
mm period: 12.8; 40 mm peroid: 10.4; liver: 24.5,
17.4 and 12.8 ml/min., respectively. After trans-
fusion, liver utilization returned to 26.3 ml/min.,
but intestinal utilization recovered only to 14.5
ml/min.

544. Method for determining plasma volume
of animals with dextran. RoBEert E. SEMPLE.
Dept. of Physiology, Queen’s Univ., Kingston,
Canada. :

A preliminary report from this laboratory
showed that a narrow high molecular weight frac-
tion of a commercial dextran solution could be
used to determine plasma volume (Proc. Canad.
Physiol. Soc., Oct., 1955). More recently a 9.4%
solution of similar dextran in saline was injected
into unanesthetized dogs (0.4-0.6 ml/kg) along
with T-1824. A protein-free extract of plasma,
secured from a single blood sample drawn 18-20
minutes after injection, was analysed for dextran
after removal of endogenous glucose by dialysis.
T-1824 distribution was determined from serial
blood samples in the usual way. In 14 experiments
the plasma volume represented by dextran was on
the average 99.3% (S.D. = 3.6%) of that meas-
ured by T-1824, indicating that the volumes of
distribution of the two substances are not signifi-
cantly different. The consistency of the method is
being tested in dogs and other animals by meas-
uring the plasma volume of an animal twice within
a short period of time. Experiments to date indi-
cate that volumes determined by dextran dilution
are reproducible to within 5%. The dextran prepa-

168

ration has certain advantages over T-1824 for
plasma volume work. Lipemia and hemolysis do
not interfere, there is no skin discoloration and the
dextran used showed a much slower and a more
consistent rate of disappearance from plasma
than did the dye. Single blood samples thus gave
results that were as satisfactory as those obtained
with serial samples after dye injection. (Supported
by Grant 9310-15, Defense Research Board of
Canada.)

545. Effects of hypothermia on pulmonary
function. J. W. SEVERINGHAUS AND M. StTupFEL
(introduced by P. O. Cuatrretp). Natl. Heart
Inst., Bethesda, Md.

Hypoventilation during hypothermia ordinarily
requires assistance to respiration. Some workers
have suggested that at low temperatures in spite
of normal rate and depth of artificial respiration,
blood pCO: may rise and pH may fall. This can
only be explained by postulating an increased dead
space or maldistribution of ventilation and blood
flow in the lung. Such other proposed causes as
changes in cardiac output, carbonic anhydrase
failure, and diffusion are not quantitatively
tenable. In 36 experiments dead space, blood and
gas distribution, compliance, blood px and gas
tensions were determined on anesthetized, cura-
rized, artificially ventilated dogs before, during
and after surface cooling to 22-26°. Anatomic dead
space (Fowler’s techinque) increased 70-90%, and
at low temperatures could not be further increased
by atropine or decreased by vagal stimulation.
Physiological dead space increased somewhat less
than anatomic dead space. The difference between
them, ‘alveolar dead space,’ primarily a measure
of the uniformity of pulmonary blood flow, was
not increased during hypothermia. Any block in
CO, excretion would have increased alveolar dead
space. Ventilation distribution (slope of alveolar
N: plateau) was unimpaired. Compliance de-
creased ng more than in controls held under anes-
thesia at 37° for the same length of time. When
tidal volume and rate were held constant during
cooling, the arterial pCO, fell at 25° to about half
of its control value, reflecting a comparable fall in
metabolism. The pu usually rose about 0.1 uv, due
to the relative overventilation, even though some
metabolic acidosis was uniformly observed.

546. Ionic transport in the sciatic nerve of the
toad, Bufo marinus. ABRAHAM M. SHANES AND
Morris D. Berman.* Nail. Inst. of Arthritis and
Metabolic Diseases, Bethesda, Md.

Sodium outflux is unaltered by oxygen lack and
by treatment with iodoacetate, dinitrophenol and
eserine, whereas potassium influx is reduced to
about one third and the outflux is increased 50%
by a combination of the first 2 experimental con-
ditions. Sodium outflux is enhanced upon return

FEDERATION PROCEEDINGS

Volume 1§

to oxygen following anoxia, and this increase ig
reduced by dinitrophenol or by a deficiency of
potassium in the medium. The augmented potas.
sium outflux during anoxia is depressed in a low
sodium medium. These and additional data sug.
gest that most of the potassium influx in vitro ig
due normally to active transport, which usually,
but not invariably, is linked with outward transfer
of sodium. During metabolic inhibition cation
permeability increases 50%, so that the loss of
potassium coupled sodium eflux is compensated
for by an increase in the ‘passive component’ of
sodium outflux; thus, the rise in fiber sodium
during inhibition is brought about by an increase
in sodium influx rather than by a decrease in out-
flux. Cocaine reduces both influx and outflux of
potassium during anoxia, and outflux in oxygen as
well, but the influx in oxygen is but slightly re-
duced; these findings are consistent with a decrease
in potassium permeability by cocaine with little
effect on the metabolism dependent transport
mechanism.

547. Resection of antral mucosa and vagec-
tomy for treatment of peptic ulcer—experi-
mental study. Danie, M. SHarrro AND Davip
V. Hasir (introduced by Haroitp G. Barker).
Dept. of Surgery, Columbia Univ., College of
Physicians and Surgeons, New York City.

The type of operation proposed for peptic ulcer
in humans continues to be varied, testifying to the
failure of achieving a standard procedure. The
results of these different operations in some pa-
tients are either recurrent ulcer, nutritional
disturbance or other complications. Since the
cause of peptic ulcer is believed to be an excess
acid formation, it is reasonable to conclude that
achieving a normal or below normal acid concen-
tration and volume should cure the condition.
Acid is formed in the stomach primarily through a
gastric phase of hormone stimulation, gastrin on
the antral mucosa and a cephalic phase through
the vagus nerves. Abolition of both phases should
result in a lowered acid production. If this could
be accomplished with maintenance of the entire
gastric pouch and normal continuity, an ideal pro-
cedure might be achieved. To produce the above,
healthy mongrel dogs were subjected to an oper-
ation removing the antral mucosa and submucosa
and suturing a tube pedicle of pars media mucosa
and submucosa to the duodenal mucosa. A py-
loromyotomy and bilateral vagectomy were also
done. The operation is technically feasible. Studies
of acid concentration before and after this oper-
ation were made.

548. The rabbit fundus oculi. HerBert SHA-
prro. Wills Eye Hosp., Philadelphia, Pa.
A nonsurgical technique has been devised for
direct observation and photography of the rabbit’s

fun
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vit!

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icy of
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ually,
ansfer
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oss of
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odium
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ly re-
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ulcer
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e the
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March 1956

fundus, including choroidal and retinal vessels.
Hitherto, study of choroidal vessels has been ap-
proached by proptosis of the eye and surgical
incision of the sclera, or exposure of the sclera,
with drying, or application of glycerine. These
techniques have the disadvantages of a) displacing
the eye out of the bony socket, 6) chemical and
surgical intervention, c) hazard of drying and cool-
ing of the tissue due to exposure, d) possibility of
overheating the eye, due to intense illumination.
All the above represent nonphysiological con-
ditions. A solid —35 diopter planoconcave contact
lens was used for the study of the retinal vessels
alone (KomnpPE 1918; LAMBERT 1934), eliminating
some difficulties inherent in (a) to (d). In the pres-
ent study, the refraction of the rabbit cornea was
neutralized by moistening the lids with Ringer’s
solution, and placing between them a glass ring
19 mm o.d., 5 mm deep, which rested on the eye-
ball. A round cover-slip is placed over the ring,
which is filled with Ringer’s solution. A liquid
neutralizing lens is thus formed, contained within
aglass shell. The choroidal and retinal vessels of
the albino rabbit may now be viewed directly in
the eye in situ without the aid of the ophthalmo-
scope and photographed. Microscopic observa-
tions can be made with a 45° mirror containing a
central aperture through which the light rays pass
into the objective. The animal’s head is immo-
bilized with a special clamp. A preliminary study
using this technique has been made of the effect of
oxygen on choroidal vessel calibre, by inserting a
tracheal cannula connected to the oxygen supply
through a suitably prepared flask.

49. Concentration of sodium, potassium and
hydrocortisone in plasma of adrenalec-
tomized and intact dogs subjected to surgi-
cal trauma. LEONARD SHARE AND HARVEY
Kri£GER (introduced by H. Gotpsiatt). Depts.
of Physiology and Surgery, Western Reserve Univ.
School of Medicine, Cleveland, Ohio.

There is a conflict of opinion as to whether or
not the adrenal cortex plays a key role in initiating
the metabolic changes which frequently occur
after trauma. There is also some question as to the
exact nature of this metabolic response. In an at-
tempt to clarify this situation, a study was made
of the response of adrenalectomized dogs, main-
tained by a constant intravenous infusion of 20
mg/day of hydrocortisone, to a moderate surgical
trauma, i.e. resection of 3-5 cm of rib under local
anesthesia. The infusion of hydrocortisone was
begun at least 5 days before an experiment. Blood
samples were taken periodically during a period
beginning 12 hr. before surgery and ending 12 hr.
after surgery. Control experiments without
surgery were also performed. The surgical trauma
produced no changes in the plasma concentrations

AMERICAN PHYSIOLOGICAL SOCIETY

169

of sodium, potassium and hydrocortisone. In a
control experiment performed on an intact dog,
there was a rise in plasma sodium concentration
and a fall in plasma potassium concentration.
These changes are attributed to the loss of blood
taken for the various analyses. Rib resection in
this same dog did not produce any additional
changes in the plasma concentration of sodium
and potassium. The plasma concentration of hy-
drocortisone was unchanged in both the control
and trauma experiments performed on the intact
dog. (Supported in part by grants from the Public
Health Service.)

550. Polarographic oxygen tension determi-
nation: factors causing calibration-change
in blood. R. H. SHeparp (introduced by R. L.
Riuey). Dept. of Environmental Medicine, Johns
Hopkins Univ., Baltimore, Md.

When a cellophane-coated, platinum oxygen
cathode is transferred from a saline calibrating
solution to blood of the same Pog, its current de-
clines to about 80% of its former value along a
simple exponential time course. This effect has
been studied using blood, blood dialysate and
different pure solutions. It is produced by many
solutes. Its magnitude for 1 solute is proportional
to the concentration of that solute. Its magnitude
for different solutes varies with the depression of
the oxygen solubility coefficient which they pro-
duce. Its time constant is essentially independent
of the concentration of solute but is proportional
to the diffusion coefficient of the solute. The effect
in blood is produced by a dialyzable, heat-stable,
trypsin-digestible substance or group of sub-
stances which diffuse at rates compatible with
molecular weights 200-300. The concentration of
this material appears to vary in different blood
samples and may be altered by procedures such as
tonometry. The ratio between the rates of diffusion
of this substance and of oxygen is about 1:40 and is
not significantly different in such different mem-
branes as cellophane, sintered glass and latex.
These findings suggest that calibration change
cannot be avoided by special electrode coatings
or special electrical circuits. More promising ap-
proaches are adjustment of solute concentration
in calibrating solutions and solving the simul-
taneous equations for the time course of the cur-
rent change. (Supported by Office of Naval Re-
search and U.S. Air Force.)

551. Blood sugar and liver glycogen levels in
irradiated and nonirradiated mice. F. G.
SHERMAN AND F. M. Dwyer.* Biology Dept.,
Brookhaven Natl. Lab., Upton, N. Y.and Dept. of
Biology, Brown Univ., Providence, R. I.

Male CF-1 mice were irradiated with 600 r of
filtered 200 kv x-rays. Nonirradiated animals re-

ceived a dummy irradiation. At }, $, 1, 3, and 6

170

hr. after irradiation, control and irradiated ani-
mals were anesthetized with Nembutal and bled
from the heart. The livers were removed immedi-
ately and frozen in liquid nitrogen. The glucose
equivalent of blood sugar, liver sugars, cold TCA
soluble glycogen and hot TCA soluble glycogen
was determined with anthrone. Irradiation re-
sulted in blood sugar elevation both in fed and
fasted animals. Small reductions in the amount of
cold TCA soluble glycogen and hot TCA soluble
glycogen were observed in irradiated fasted ani-
mals. A corresponding increase in liver sugars was
found in these animals. A large increase in the
cold TCA soluble glycogen and free sugar fraction
was observed in the livers of fed irradiated ani-
mals. Peak values were obtained 30-60 min. after
irradiation. Six hours after irradiation the liver
free sugar and cold acid soluble glycogen values
had returned to control levels in the fed animal
experiments. (Research carried out at Brook-
haven Natl. Lab. under the auspices of the U. 8.
Atomic Energy Commission, and supported in
part by a grant-in-aid to Brown Univ. from the
American Cancer Society upon recommendation
of the Committee on Growth of the Natl. Re-
search Council.)

552. Sex of infant in relation to nuclear mor-
phology of cells in human amniotic fluid.
LanprRuM B. SHetties. Dept. of Obstetrics and
Gynecology, College of Physicians and Surgeons,
Columbia Univ., New York City.

The presence or absence of a sex chromatin
mass in intermitotic nuclei of cells of amniotic
fluids was studied in relation to sex of offspring.
Ten cubic centimeters of amniotic fluid was ob-
tained at delivery by low-cervical cesarean section
or vaginally in 20 patients with female and 20 with
male infants. The fluid was centrifuged, the super-
natant portion discarded, the remaining cells
smeareg on slides and while still moist immersed
in modified Davidson’s solution for 3-24 hr., after
which they were hydrated and stained according
to a modified Feulgen technique, with a light
green counterstain. Final observations were made
under oil immersion. The degree of variation in
morphology of the cells in a given amniotic fluid
reflected the various possible sources of their ori-
gin: skin, gastrointestinal, respiratory, genitouri-
nary tracts, umbilical cord, amnion. In cells in sat-
isfactory condition for study from amniotic fluids
which contained female infants, 28-65% of the
nuclei in different samples contained the charac-
teristic Feulgen-positive, planoconvex, rounded or
flattened sex chromatin body lying against the
inner surface of the nuclear membrane. In none of
the nuclei of cells from fluids in which males de-
veloped was a peripheral sex chromatin mass seen,
regardless of how the nucleus was rolled. With

FEDERATION PROCEEDINGS

Volume

careful study of a properly prepared and stained
slide of cells from amniotic fluid, the sex of the
infant should be ascertained with very little
chance of error.

553. Effects of intravenous administration of
fat emulsion on blood coagulation in dogs,
Harrison H. SHouupers, Jr., Ropert C. Harr.
MANN AND H. C. MEnG (introduced by PR.
Haun). Depts. of Physiology and Medicine,
Vanderbilt Univ. Med. School, Nashville, Tenn.
This work was designed to determine the effects

of intravenous administration of fat emulsions on
blood coagulation. A series of 30 dogs under
Nembutal anesthesia were used. All blood samples
were drawn from the external jugular vein into
silicone-coated syringes. A 30% cottonseed oil
emulsion was introduced into the saphenous vein
by motor-driven pump at 0.06 ml/kg/min. for %
min. Within 10 min. after beginning the infusion
the following changes were found: 1) a sharp drop
in platelet count to as low as 5000/cu mm; 2) a
prolonged bleeding time, increased capillary
fragility and prolonged clot retraction time; 3) a
shortened coagulation time from a normal of 20 to
less than 5 min; 4) elevation of hematocrit. No
significant change in plasma fibrinogen concen-
tration, thrombin clotting time, prothrombin time
or ‘prothrombin consumption’ was observed. In
vitro experiments showed that the emulsion with
concentrations of 0.5-5.0 gm% had no effect upon
coagulation time, prothrombin time, clot retraec-
tion time or thrombin clotting time. Subsequent in-
fusions of emulsion within 2 wk. were followed by
a markedly prolonged coagulation time in some
animals. No evidence of circulating anticoagulant
or procoagulant was detected. Dog chyle adminis-
tered intravenously at 0.6 ml/kg/min. for 10 min.
produced a decrease in coagulation time but no
change in platelet count; however, injection at a
rate of 1.8 ml/kg/min. was followed also by pro-
nounced thrombocytopenia. It was found that
certain nonionic emulsifying agents alone pro-
duced changes similar to those observed in dogs
receiving an infusion of cottonseed oil emulsion or
dog chyle.

554. Incorporation of labeled glucose into the
DPN of mouse liver. Louis SHusTER* AND
ABRAHAM Go.pIN. Lab. of Chemical Pharma-
cology, Natl. Cancer Inst., Bethesda, Md.
Kaplan et al. (Science 120: 437, 1954) have re-

ported an eightfold increase in the DPN content

of mouse liver following the injection of nicotina-
mide. In an attempt to trace the source of the
ribose moiety of this newly synthesized DPN,
labeled glucose was used. Each of 10 mice was in-
jected intraperitoneally with 500 mg/kg of nico-
tinamide and 5 we (2.5 mg) of C'-labeled glucose.
The mice were killed after 7 hr. and DPN was iso-

‘ume 15

stained
of the
- little

ion of
| dogs,
Hart.
PAP.
icine,
"enn.
effects
ions on
under
amples
in into
ed oil
18 vein
for 20
ifusion
'p drop
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‘it. No
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ed. In
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ved by
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March 1956

lated from the livers by ion-exchange chromatog-
raphy. The specific activity of DPN obtained
after injection of uniformly labeled glucose was
2,750 emp/uM, while that from C-labeled glucose
was 2,430 cpm/um. The DPN was hydrolyzed with
snake venom pyrophosphatase and the resulting
mononucleotides were separated by ion-exchange
chromatography. In both cases the specific ac-
tivity of the nicotinamide mononucleotide was ap-
proximately twice that of the adenylic acid.
Distribution of C'4 in the free ribose obtained by
enzymatic degradation of the mononucleotides is
being investigated. Since it would involve the loss
of label in carbon 1, the oxidative decarboxylation
of glucose to ribose does not seem to be the main
pathway of pentose biosynthesis in this case. The
transketolase-transaldolase mechanism, by which
carbons 1 and 2 of glucose are both incorporated
into pentose, could provide a better explanation of
the observed results.

555. Orosomucoid and protein content of
nasal polyps. S. SILBERBERG* AND H.R. Catcu-
poLE. Dept. of Pathology, Univ. of Illinois College
of Medicine, Chicago.

Nasal polyps excised from human patients were
frozen in liquid air and dried in vacuo. The dried
tissues were sliced thin and defatted with
methanol-chloroform, acetone and ether. Weighed
amounts of the dried, fat-free tissues were ex-
tracted with normal saline solution so that each
ce represented 30 mg dry tissue. Total protein was
determined on aliquots by the Lowry method. A
quantitative immunochemical method was used
for orosomucoid: one-tenth ml of the polyp extract
in 8% NaCl was overlaid with 1.4 ml diluted
chicken antihuman orosomucoid serum, also in
8% NaCl. Following incubation at 38°C for 10
min., the turbidity developed was read at 450 my
ina Coleman spectrophotometer, and the concen-
tration of the unknown read from a standard
curve. In 3 polyp samples, orosomucoid repre-
sented 10-25% of the total protein of the extract.
The corresponding value for orosomucoid in blood
serum obtained by similar methods is approxi-
mately 0.2%. The results point to the presence in
these polyp tissues of a mucoprotein having the
same antigenic properties as blood orosomucoid,
in such relative proportions as to rule out the
likelihood of it being part of a serum exudate. (We
are indebted to Dr. Max Samter, Allergy Unit,
Univ. of Illinois, for material used in this study.)

6. Glucose tolerance and the response to
insulin in young and old subjects. FELIx
A. SILVERSTONE,* MARTIN BRANDFONBRENER,*
NaTHan W. SHock AND Marvin J. YIENGST.*
Section onGerontology, Natl. Heart Inst., Bethesda,
and Baltimore City Hosps., Baltimore, Md.

A glucose tolerance test was compared with the

| wae tne

AMERICAN PHYSIOLOGICAL SOCIETY

171

same procedure with the simultaneous adminis-
tration of insulin. When both tests are performed
on the same subject, an estimate of both carbo-
hydrate tolerance and response to insulin is pos-
sible. Decreased glucose tolerance has been re-
ported in old people but insufficient study has been
directed at the possible mechanisms involved. If
reduced tolerance is equivalent to reduced cell
uptake of sugar, to what extent is the supply or
responsiveness to insulin involved? Thirty-five
carefully screened male subjects, 23-86 yr. of age,
were given an intravenous glucose tolerance test
(GTT) and glucose-insulin tolerance test (GITT),
using 50 ml of 50% glucose and 5 units of HGF
insulin/mM? of body surface on different days.
Venous blood was sampled at 5-min. intervals
and analyzed by the Nelson-Somogyi method.
Analyses of the blood glucose concentrations
between 10 and 60 min. after administration in-
cluded arithmetic curves and rate measurements
(K). The latter were determined by least squares
fits to plots of log glucose concentration against
time; the relationships were linear. Insulin re-
sponse was taken as the difference (AK) between
K for the GTT and K for the GITT. Significant
age differences were found in the rates of
disappearance of glucose from the blood plasma
both with and without insulin. Insulin had a sig-
nificantly greater effect in young subjects than
in old.

557. Effect of pneumothorax on pulmonary
circulation in normal and vagotomized
dogs. DANIEL H. Stmmons* AND ALLAN HEMING-
way. Depts. of Physiology and Medicine, Univ.
of California Med. Ctr. and VA Ctr., Los Angeles.
Pulmonary artery and pulmonary vein pressures

were measured by fluoroscopically placed cardiac

catheters and strain gauges and cardiac output by
the Fick method on 12 normal, anesthetized,
tracheotomized dogs and on 9 dogs with bilateral
cervical vagotomy. Measurements were made
before and after inducing a pneumothorax with

25 ml/kg. of air (approximately equal to the

normal functional residual capacity). From these

data, the pulmonary pressure gradient and pul-
monary vascular resistance were calculated. In
the case of normal dogs, pulmonary artery pres-

sure rose gradually after pneumothorax to 125%

of control values between 2 and 3 hr. later. Pul-

monary artery pressure rose more rapidly in

vagotomized dogs to 113% of control values in 30

min. Changes in pulmonary venous pressures of

normal dogs paralleled changes in pulmonary
arterial pressures. In vagotomized dogs, however,
the increases in pulmonary vencus pressure were
significantly greater than changes in pulmonary
artery pressure, reading 139% of control values in
30 min. In neither group of dogs did the pulmonary

172

artery-pulmonary vein pressure gradient change
significantly. Both greups exhibited a marked
drop in cardiac output. Values for normals aver-
aged 55% of control values between $ and 23 hr.
after pneumothorax, while values for vagotomized
dogs averaged 69% of control values } hr. after
pneumothorax. As a result, pulmonary vascular
resistance increased to 190% and 171% of control
values in the 2 groups.

558. Effect of age on mean spatial QRS and T
vectors. ERNst SIMONSON AND ANCEL Keys.
Lab. of Physiological Hygiene, Univ. of Minne-
sota, Minneapolis.

Mean spatial QRS and T vectors, constructed
from the conventional electrocardiogram by
means of a mechanical analyzer, were compared
in 105 young men from 18 to 28 yr. and 178 older
men in the 6th decade. All items of vector analysis
(magnitude, azimuth, elevation, of the mean
QRS and T vectors, and the angle between these
vectors) showed highly significant differences
between the group means. In the older men, both
the QRS and T vector were rotated more an-
teriorly (larger azimuth angle), and were more
elevated, their magnitude was smaller, and the
angle between the vectors was larger, as compared
to the younger men. The change of the elevation
angle corresponded to a left shift of the QRS and
T axes, and the change of the magnitude corre-
sponded to a smaller QRS and T amplitude in the
conventional scalar ECG leads with age. The
range of interindividual variability was signifi-
cantly larger in the older men for the elevation
angles of the QRS and the T vector. Preliminary
age-corrected limits are presented, as calculated
from the standard deviation, for 95% normal male
population. The age differences between the
upper normal limits of azimuth and elevation are
larger than those between the lower normal limits.

559. Temperature dependence of narcosis in
nerve. RaymMonp A. Ssop1n (introduced by
L. J. Muuurns). Biophysical Lab., Purdue Univ.,
Lafayette, Ind.

The degree of conduction block observed in frog
nerve at a given concentration of narcotic has
been shown tovary markedly within a temperature
range not affecting conduction. Using an ap-
paratus designed to maintain a localized nerve
block at any temperature desired, it has been
possible to demonstrate that blocking is
diminished at higher temperatures when a given
activity of Novocaine or cocaine is employed. The
effect is best observed at a drug activity which
results in about a 30% block at 20°C. Under these
conditions, a plot of the percentage block ob-
served, at constant drug activity, versus the
temperature, yields a curve with fairly uniform
negative slope in the range between 10° and 35°C.

FEDERATION PROCEEDINGS

Volume 1§

If the blocked region is maintained at the lower
temperature initially, it is possible to diminish or
even remove the block by raising the temperature,
If the block is at the higher temperature initially,
it can be augmented by lowering the temperature,
These effects are very rapid, occurring immedi-
ately when the temperature is changed. Since
blocking by drug action is a fairly slow process,
15-20 min. being required to approach an equi-
librium, the observations suggest that the parti-
tioning of these drugs to the nerve membrane and
their narcotic action may be 2 distinct processes,
Similar results are observed with 1,3,5-trimethyl-
benzene (mesitylene) if the block is less than
40%. More severe blocks with mesitylene become
accentuated by either raising or lowering the
temperature from a critical value of about 27°C,
A change of 2°C produces as much as a 20% in-
crease in block.

560. Effects of lysergic acid diethylamide and
related amines on the electrical activity of
the rat brain. ALAN G. SLocoMBE (introduced
by Hupson Hoaguanp). Worcester Fndn. for
Exptl. Biology, Shrewsbury, Mass.

Lysergic acid diethylamide (LSD) and adreno-
chrome have been reported to produce psychotic
experiences in normal human subjects. Dis-
turbances in adrenalin and serotonin metabolism
have each been suggested as possibly involved in
psychotic behavior. This study was undertaken to
compare the actions of these psychosomimetic
drugs on the central nervous system with those
of serotonin, epinephrine, and nor-epinephrine.
Spontaneous and evoked electrical potentials were
used as indications of neutral activity. The ex-
periments were done on albino rats, anesthetized
with either Pentothal or ether, or on unanes-
thetized rats the spinal cords of which had been
sectioned previously. It was found that under
Pentothal, all of these drugs caused a flattening of
spontaneous activity both at cortical and at a
variety of subcortical sites. Inhibition of the
transcallosal potential was found to parallel
diminishing spontaneous activity with serotonin.
In animals anesthetized with ether and in unanes-
thetized spinal animals there was no depression of
spontaneous activity, but in some cases an
increase in slow wave amplitude superimposed on
an unchanged fast activity. There was no apparent
qualitative difference between the effect of these
drugs. Quantitatively, however, they differed
from one another in the dose required to depress
spontaneous activity in Pentothal anesthetized
animals. Their effectiveness may be ranked in
the order: serotonin, LSD, epinephrine, nor-
epinephrine, and adrenochrome. It is concluded
that an apparent synergy exists between these
drugs and Pentothal.

Mar

ume 1§

lower
ish or
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March 1956

561. Effect of hypothalamic lesions on adrenal
ascorbic acid response to stress in the male
rat. Marcaret A. SiusHeR* aNnp SIDNEY
Roserts. Depts. of Anatomy and Physiological
Chemistry, School of Medicine, Univ. of Cali-
fornia Med. Ctr., Los Angeles.

The effect of hypothalamic lesions on the re-
sponse to the stress of laparotomy has been studies
in the adult male Sprague-Dawley rat. Electro-
lytic lesions were made bilaterally using a 2-ma
current for a duration of 10 sec. The response to
stress was measured by the change in adrenal
ascorbic acid (Roz aND KevuTuer, 1947) 1 hr.
postlaparotomy. In 10 rats with lesions involving
only the tuber cinereum and anterior border of
the mammillary bodies a complete block of the
stress response was noted. Although daily water
intakes over 135 ml were noted in all rats blocked
to stress, other rats with daily water intakes as
high as 250 ml showed normal stress responses. In
both groups the hypothalamic-hypophyseal tract
and median eminence remained intact. Gonado-
tropin function appeared unaltered in all rats
blocked to stress on the basis of weights and
histology of the testes, seminal vesicles and
prostate as well as acid phosphatase determina-
tions of the prostate. It is concluded that hypo-
thalamic lesions in the tuber cinereum and
anterior border of the mammillary bodies pro-
duced a complete block in adrenal ascorbic acid
response to operative stress. Furthermore, these
blocks occurred in rats with high water intake
although the hypothalamic-hypophyseal tract and
median eminence remained intact.

562. Effect of partial body shielding during
irradiation on antibody titers in mice.
Fatconer SmitH, H. Jeanette Rutu* anp
Marie M. Grenan.* Natl. Cancer Inst.,
Bethesda, Md.

Peak anti-sheep erythrocyte hemolysin titers
were compared in N.I.H. mice exposed over the
whole body to 85, 290, 360 and 450 r 200 kvp x-rays
under Nembutal anaesthesia. Littermate mice
were given Nembutal and comparable doses of
tadiation through }” perforated lead plates, per-
nitting various fractions of the body to be
imadiated. Treatment groups containing 10-20
mice were immunized and 50% hemolytic end
points were obtained for pooled serum samples.
At 1 and 2 wk. after exposure to 85 r to the whole
body the titers were 1:326 and 1:433, respectively,
while in mice exposed to this amount of radiation
oer 10% of the body the titers were 1:424 and
1:435 compared to 1:537 in nonirradiated mice.
The immune response was abolished until the
{th wk. after 450 r to the whole body but in mice
exposed to 450 r to 10% of the body the titers were
1:188 at 1 week after irradiation and gradually

AMERICAN PHYSIOLOGICAL SOCIETY

173

Increased thereafter. Whole body exposure of
mice to intermediate doses, 290 and 360 r,
abolished antibody production until the 3rd wk.
while a reduced immune response was observed at
1 wk. after irradiation in mice exposed to these
doses over 41% or 55% of the body. The results
of these experiments show that the degree of
impairment of the hemolysin response in mice
increases with radiation dose. Damage to the
production of hemolysin appears to be directly
related to the amount of the total body which is
irradiated.

563. Production of secondary shock by
a combined bleeding-aortic occlusion
method. James J. SmitH, Ropert A. GRACE
AND Donato J. Kerstina (introduced by
Rosert D. Tayrtor). Dept. of Physiology,
Marquette Univ. School of Medicine, Milwaukee,
Wisc.

Fatalities in aortic occlusion or tourniquet shock
depend mainly on occlusion time and the amount
of ischemic tissue involved. To study these vari-
ables further, intra-aortic balloon occlusion was
produced in morphinized-barbitalized dogs.
Total and near-total occlusion of the lower
thoracic aorta for 14-2} hr. resulted in the death
of 20%-100% of animals depending on the degree
and length of time of the occlusion. The variable
but pronounced central hypertension proved
however to be an undesirable complication. In
another series a partial occlusion of the thoracic
aorta (T8-9) was combined with bleeding from a
carotid artery into an inverted pressure reservoir
containing anticoagulant. Thus blood pressure
above and below the occlusion could be inde-
pendently predetermined and controlled. 80 and
30 mm Hg mean pressure levels were maintained
in the upper and lower body segments, respec-
tively, for 2-3 hr. Maximum bleeding volumes
averaged 47.4 ml/kg. After reinfusion and balloon
release the blood pressure generally returned to
control levels and then gradually declined. Mor-
tality ranged from 55 to 80% depending on occlu-
sion time. In control groups with equivalent bled
volumes but no occlusion, mortality was below
10%. By permitting control of arterial perfusion
pressures in different body areas, such a method
may be of value in the study of secondary shock.

564. Action of MK-57 upon the fetus and upon
labor. FRANKLIN F. SnypER. Depts. of Obstetrics
and Anatomy, Harvard Univ., Boston, Mass.
MK-57 (6-methyl-A*-desoxymorphine) is of

interest for obstetric use by reason of analgesic

potency greater than that of morphine but of
definitely shorter duration of action. As a guide to
clinical use, determinations have been undertaken
in rabbits of the effect of MK-57 upon the fetus

174

before birth and upon the labor mechanism. By
direct observation of full-term rabbit fetuses
showing rhythmical respiratory movements within
the unopened uterus following laparotomy in a
saline bath, it was found that injection of MK-57
0.1 mg/kg i.v. into the mother promptly decreased
the fetal respiratory rate below half that occurring
before injection. Regarding the action of MK-57
upon the labor mechanism, no increase in the
average incidence of stillbirths or of prolonged
labor was noted following injection at the time of
labor, 30 or 31 days, of 2 doses of MK-57 1.3 mg/kg
each, given 1.M. at intervals of 2 or 4 hr. In 461
births fetal mortality was 8%. However, following
4 doses of MK-57 or a total of 5.2 mg/kg, the
incidence of stillbirths increased, averaging 20%
in 323 births, or more than twice that of uninjected
controls. (Aided by a Public Health Service re-
search grant.)

565. Effects of lysergic acid diethylamide on
cerebral circulation and metabolism in
Louts Soko.Lorr, SEyMouR PERLIN,*

man.

Conan KorNETSKY* AND Seymour S. Kerry.
Natl. Inst. of Mental Health, N.I.H., Be-
thesda, Md.

Lysergic acid diethylamide has recently been
found to produce in minute doses marked aberra-
tions of psychological and mental functions.
Because these disturbances simulate those ob-
served in naturally occurring psychoses, there
has been considerable interest in the mechanism
of action of the drug. The results of in vitro studies
on its effect on cerebral metabolism have suggested
possible alterations in cerebral oxygen utiliza-
tion. Also, its reported in vitro antagonism to the
effects of serotonin has led to speculation con-
cerning its effects on cerebral circulation. In
order to determine whether similar effects occur
in vivo in man during the actual presence of drug-
induced psychosis, studies of the effects of LSD on
cerebra¥ blood flow and metabolism were per-
formed in 10 normal subjects and 8 schizophrenic
patients by means of the nitrous oxide method.
Each study consisted of a control determination
followed by another performed during the psycho-
sis produced by the intravenous injection of
approximately 120 ug of LSD. Despite the clearly
altered status of mental and psychological func-
tions, no changes in cerebral blood flow, vascular
resistance, oxygen consumption, glucose utiliza-
tion, orrespiratory quotient were observed. Small
but statistically significant increases in mean
arterial blood pressure and arterial hemoglobin
concentration were observed. It appears, then,
that in vivo the psychological alterations pro-
duced by LSD are not associated with over-all
changes in cerebral circulation or in gross oxygen
and glucose utilization by the brain.

FEDERATION PROCEEDINGS

Volume 15

566. Effect of cardiac glycosides on K trans.
port in human red cells. A. K. Sotomoy,
Tuomas J. Gruu IIL* anp G. LENNARD Go1p,*
Biophysical Lab., Harvard Med. School, Boston,
Mass.

Schatzmann (Helv. physiol. Acta 11: 346, 1953)
has shown that cardiac glycosides inhibit the
uptake of K by cold-stored red cells; his results
have been extended with the use of K* by Joyce
and Weatherall (J. Physiol. 127: 33 p, 1955) and
Glynn (J. Physiol. 128: 56 p, 1955). The present
studies are concerned with the kinetics of cation
transport in human red cells incubated in vitro
at 37°C. Analogue computer analysis of K influx
data, measured by K*, indicates that cardiac
glycosides inhibit the first step of K entrance. A
dose response curve was obtained with ouabain
over the concentration range 10~* to 107° m/l.
About 25% of the K influx is unaffected by
ouabain; over the sensitive range, 1077-5 molar
ouabain produces half inhibition. Glucose utiliza-
tion is unchanged. Kinetic analysis of influx is
not possible if a single molecule of glycoside
reacts with a single site or molecule on the cell
surface; instead, two or more sites are required,
Ouabain and other glycosides have been classified
according to the attachment constant, K,, the
equilibrium constant between free glycoside and
glycoside bound to the cell, and these values are
compared with the mean lethal dose in cats.
Ouabain also causes the cell to gain Na rapidly.
The kinetics that describe the transport of Na in
normal cells do not suffice to describe the ouabain
poisoned system, and it appears that another
compartment may be necessary to account for
glycoside action.

567. Transtubular electrical potentials of the
rat kidney. StipNEY SoLomon (introduced by
E. Fiscurr). Dept. of Physiology, Medical College
of Virginia, Richmond.

By means of microelectrodes it is possible to
measure the electrochemical potential difference
(p.d.) across the surface tubules of the rat kidney.
Measurements on randomly selected tubules result
in a bimodal population distribution of p.d. with
means of the lower value portion of the distribu-
tion ranging from 19 to 39 mv, and means of the
higher values ranging from 34 to 70 mv. In all
cases, the inside of the tubule is negative to the
outside. Decapsulation of the kidney is without
effect on the p.d. values obtained. The p.d. can
be maintained for extended periods of time. It
is reduced in magnitude both by interrupting cir-
culation and by treatment with the mercurial
diuretic, Mercuzanthin. In untreated animals
the magnitude of the p.d. is inversely related to
the rate of urine flow. By using phenol red to
identify proximal and distal tubules it appears

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March 1956

that the low values come from proximal tubules,
whereas the high values are derived from distal
tubules. Phenol-red treatment causes a fall in
transtubular p.d. in some experiments.

568. Local cerebral blood flow in spreading
cortical depression. R. R. SONNENSCHEIN
AND R. M. WaLKEr.* Dept. of Physiology, Univ.
of California Med. Ctr., Los Angeles.

Local blood flow in the outer 2 mm of the brains
of cats and rabbits anesthetized with Dial was
observed by the thermoelectric method described
by Sonnenschein et al. (Am. J. Physiol. 183: 000,
1955). In some experiments, cortical temperature
alone was used as the index of blood flow. Attempts
to produce spreading depression were made by
electrical stimulation of the cortex and by applica-
tion of 1% KCI to limited areas; the latter method
was more successful. Depression, indicated by
electrocorticograms, was more readily produced
in the rabbit than in the cat. Electrical stimula-
tion (bipolar, biphasic, 50 cps, 2-10 v) usually
led to an immediate increase in local blood flow
(often associated with a transient fall in arterial
pressure), lasting } to 1 min. After a further latent
period of up to 4 min., varying with the distance
between stimulus and site of blood flow detection,
a second prolonged rise occurred, lasting 4-10
min. When depression occurred, it was closely
correlated with the delayed alteration in blood
flow. Occasionally, blood flow changed without
obvious depression. Application of KCl was fol-
lowed only by the delayed increase in blood flow,
again closely correlated with depression. In a
quarter of the observations, a decrease in blood
flow was observed. (Supported by contract with
Aero-Med. Lab., Wright-Patterson AFB, Ohio.)

69. Distribution and nature of antibodies
against horse serum produced in rabbits,
rats and guinea pigs. Irvina L. Spar,*
Wituram F. Bate, Dotores E. Wouire* anp
Witu1am Dewey.* Dept. of Radiation Biology,
Univ. of Rochester School of Medicine, Rochester,
WY.

Rabbits were immunized against horse serum by
periodic subcutaneous injections until well de-
veloped Arthus-type reactions resulted. Several
days after the last injection the animals were
killed by saline perfusion, and serum or plasma,
skin from various sites, and other organs titrated
for antigen-binding or -precipitating ability using
I'!labeled horse serum protein. Skin from non-
injection sites and perfused organs had antigen-
binding capacity ranging up to 20-30% that of
antigen precipitated by the same weight of serum
from the same animal. This antibody was all, or
Nearly all, extractable from skin minced with
scissors by shaking with buffered saline at 37°C

AMERICAN PHYSIOLOGICAL SOCIETY

175

for 1 hr. No tissue-fixed antibodies that are some-
times postulated as a factor in defense against
disease were demonstrated in these experiments.
Skin from areas near, but not including, recent
injection sites had antigen-binding capacity equal
to or greater than that of the same weight of
serum. Lymph nodes near the injection site had
antigen-binding capacity several times greater
than mesenteric lymph nodes of the same im-
munized animal. It was not possible to demon-
strate in skin or serum of rats or guinea pigs
subjected to the same immunizing procedures
any tendency to precipitate or bind I'*!-labeled
horse-serum proteins greater than in control
nonimmunized animals. Presumably antibodies
present in these species were neither of a precipi-
tating or a tissue-fixed type. (Supported by the
Atomic Energy Commission.)

570. Radiostrontium metabolism in man.
Herta Spencer, Eva BerGerR AND DANIEL
LaszLo (introduced by Eugene Y. Brrcer).
Div. of Neoplastic Diseases, Montefiore Hosp.,
New York City.

The metabolism of strontium is of theoretical
and of considerable practical interest since
strontium” is a major hazard of atomic fission.
No information is currently available on its
metabolism in man. The metabolism of radio-
strontium was investigated in 10 patients using
carrier-free Sr®, a pure y-emitter with a 65-day
half-life. The tracer was administered intra-
venously and/or orally. Frequent specimens of
plasma and urine were assayed in the early phase
of the study. Daily plasma samples, 24-hr. urine
collections and all fecal specimens were analyzed
thereafter. After the intravenous injection of the
isotope the plasma level of Sr® declines rapidly
and the major excretion of the isotope occurs
through the kidney. After the ingestion of
strontium the plasma level and the urinary excre-
tions rise gradually and the major portion of the
isotope passes unabsorbed through the intestine.
Excellent agreement was found between the
metabolism of the intravenously injected
strontium and the absorbed fraction of the orally
administered tracer.. The marked differences in
Sr®§ metabolism correlated well with the differ-
ences of the skeletal state of the patients studied.
Intravenously administered calcium was used as
the ‘carrier’ to increase strontium excretion and
marked enhancement of urinary strontium was
noted in all cases. Examples of the metabolism of
intravenously and of orally administered
strontium, of the enhancement of strontium ex-
cretion by calcium and a comparison of the
metabolism of radiocalcium and of radiostrontium
studied in the same patients will be presented.

176

571. Phasic flow relationships in the aorta.
MERRILL P. SPENCER AND ADAM B. DENISON JR.
Dept. of Physiology and Pharmacology, Bowman
Gray School of Medicine, Winston-Salem, N.C.
The phasic flows (FP) at various points along

the dog’s descending aorta were recorded with a
240~ square-wave electromagnetic flowmeter
using magnets of sufficient size to permit only 20%
compression of one diameter. Simultaneously, the
differential pressure (AP) across each point was
recorded by needle punctures. The results demon-
strate the aorta to be an elastic and almost fric-
tionless tube through which the flow is conducted
as a wave motion superimposed on a net forward
movement. The AP pulse accelerates the blood
most when the gradient early in systole is greatest
in the direction of mean flow and decelerated
most when the gradient is greatest (usually at the
time of the incisura) against the direction of flow.
This accounts for nearly 90° of phase shift between
the AP and the FP contours. Backflow was found
frequently in the normal thoracic and abdominal
aorta, though more frequently in the latter. When
the aorta is gradually occluded distal to the
flowmeter, the pressure gradient reverses earlier
in systole and the FP shows correspondingly
earlier deceleration and more prominent backflow.
The FP then resembles those obtained by others
with AP flowmeters with peak flow in early systole
and backflow at the time of the incisura. When
resistant flow is produced by application of small
magnets, the AP and FP contours approach that of
the central pulse and follow the phase and wave
form of the applied pressure. Backflow disappears
as the mean flow decreases.

572. Activation of the striopallidum by
diencephalic impulses. E. A. Sprecet, E. G.
SzEKELY* anD W. W. Baxer.* Dept. of Ezpil.
Neurology, Temple Univ. School of Medicine,
and Dgpt. of Pharmacology, Jefferson Med. Col-
lege, Philadelphia, Pa.

In an analysis of the mechanisms responsible
for the cessation of involuntary movements of
extrapyramidal origin in sleep and their increase
by emotional excitement, it was sought to deter-
mine from which part of the diencephalon the
striopallidum receives afferent impulses. In cats
3% strychnine solution or penicillin in oil was
injected in various parts of the thalamus, or
electric stimulation with single or repeated shocks
was performed. The effects upon the electric ac-
tivity of the caudate nucleus and the pallidum
were recorded. The experiments indicate a multi-
ple inflow of impulses from the thalamus to the
striatum and/or pallidum, particularly from
parts of the so-called nonspecific diffuse thalamic
projection system (nucleus ventralis anterior,
intralaminar nuclei, centrum medianum). The

FEDERATION PROCEEDINGS

Volume 1§

effects obtained from association nuclei, e.g. the
dorsomedial nuclei, are rather irregular and may
be due partly to stimulation of adjoining intra.
laminar or paraventricular cell groups, partly to
excitation of collaterals of corticopetal fibers,
Stimulation, particularly of the nucleus ventralis
anterior and the intralaminar nuclei, with single
shocks may elicit, after latent periods up to over
100 usec., chiefly in the caudate nucleus, slow
waves and trains of 6-11/sec. waves that may
replace the spontaneous rhythm. In view of the
important role which the diffuse nonspecific pro-
jection system plays in maintaining consciousness,
these findings may help one to understand the
parallelism between level of consciousness and
intensity of involuntary movements of extra-
pyramidal origin. (Aided by Grant B-470, Natl.
Inst. of Neurological Diseases and Blindness.)

573. Effect of heparin, protamine, alimentary
lipemia and fasting on the plasma unesteri-
fied fatty-acid level. JoHn J. SPITZER AND
Harvey I. Mituer.* Div. of Labs. and Research,
New York State Dept. of Health, Albany.
Protein-bound, unesterified fatty acids of

plasma (UFA) provide one of the main transport

mechanisms for lipids. The metabolic importance
of UFA was investigated in dogs. Injection of
heparin into lipemic dogs produced the well-known
elevation of UFA simultaneously with the ap-
pearance of clearing factor in the blood. Injection
of protamine sulphate reduced the elevated UFA
level. During active fat absorption, the UFA
concentration increased. This rise was usually
somewhat delayed as compared to the course of
lipemia. In lipemic animals, an arteriovenous UFA
difference was frequently encountered in the
femoral vessels indicating the use of this route for
fat removal. Forty-eight to 72 hr. of fasting caused

a marked elevation in UFA level. On the basis of

the above findings, it seems that the injection of

heparin or protamine alters lipid transport by
influencing both main vehicles, lipoproteins and

UFA. Transport, during fat absorption, utilizes

lipoproteins primarily but also UFA to a sig-

nificant degree. During fasting, dogs appear to
use UFA as the prevailing transport mechanism.

574. Site of spinal monosynaptic inhibition.
James M. Spracus. Rockefeller Inst. for Med.
Research, New York City, and Dept. of Anatomy,
School of Medicine, Univ. of Pennsylvania,
Philadelphia.

Test monosynaptic reflex discharges evoked and
recorded in sacral 1 dorsal and ventral roots re-
spectively are facilitated by conditioning of
ipsilateral lumbar 7 dorsal root and inhibited by
lumbar 6 dorsal root. Similarly, L6 facilitates L7,
and L5 inhibits L7 and/or Sl monosynaptic

515

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March 1956

reflexes. Test monosynaptic reflexes in S2 and S3
are inhibited by conditioning contralateral S2
dorsal root (LLorEp, 1941, 1944). Only effects with
latency of 0.5 msec. or less are considered. The
inhibition appears to be uncontaminated by
facilitation. Lumbosacral dorsal roots were sec-
tioned individually in 6 cats, and degenerating
collaterals were traced by a modification of the
Nauta silver technique. This method selectively
stains degenerating axon terminals up to the
membrane of the postsynaptic neuron, but usually
not the bouton itself. Serial sections prepared by
the Nauta technique, supplemented by others in
Nissl, were plotted. The motor nuclei were later
identified by reference to Romanes (1951). Those
dorsal root collaterals which evoke only inhibitory
effects on test monosynaptic reflexes, as outlined
above, do not reach the cell bodies of the
motoneurons but terminate on their dendrites.
Those collaterals which lie in areas of mono-
synaptic facilitation lie on both cell bodies and
dendrites of these motoneurons. The inhibitory
terminals are considerably fewer in number than
are those found in facilitatory areas. These find-
ings that the cellular sites of action of spinal
monosynaptic inhibition and facilitation are
different should not be extended to other neurons
in other parts of the CNS without considerable
caution. (Supported by PHS grant B-460(C2).)

315. Effect of chlorpromazine on _ survival
from hemorrhagic shock. G. B. Spurr,*
Enrp ALLBAUGH FARRAND* AND STEVEN M.
Horvatu. Dept. of Physiology and Cardiovascular
Lab., State Univ. of Iowa, Iowa City.
Hemorrhagic shock was produded in 54 adult

mongrel dogs by reducing the mean arterial blood
pressure to 30-35 mm Hg and maintaining this
level for 1 hr. without subsequent reinfusion of the
shed blood. Sixteen animals served as untreated
controls. Six per cent of these dogs survived the
procedure. Administration of 2 mg/kg of chlor-
promazine 30 min. previous to the onset of hemor-
thage resulted in a 50% survival which was
significantly increased over the control group. No
such protective action was observed when chlor-
promazine was given in a dose of 5 mg/kg 30 min.
before hemorrhage or when 2 mg/kg was given
Shr. before or immediately after hemorrhage.
The results indicate that the protective action of
the 2 mg/kg dose of chlorpromazine 30 min. pre-
vious to hemorrhage was not related to the
autonomic blocking properties of the drug. The
mechanism of this beneficial action is not clear
at present.

316. Protection in hemorrhagic shock by
N - ethyl - N - hexahydrobenzy! - 8 - chloro-
ethylamine (G-D 131) given after initiation

AMERICAN PHYSIOLOGICAL SOCIETY

177

of bleeding. S. G. Srrxant1a,* Srtvio BagEz,

ANNE CARLETON* AND EPHRAIM SHoRR.! Cornell

Univ. Med. College, New York City.

It is the consensus that agents (antibiotics,
neurogenic blocking drugs) so far found protective
against hemorrhagic or traumatic shock must be
administered prior to induction of shock syn-
drome. Our recent studies, reported elsewhere,
have shown that N-ethyl-N-hexahydrobenzyl-s-
chloroethylamine (G-D 131) is also highly protec-
tive when given prior to shock. This tertiary
amine, chemically related to Dibenamine series of
adrenergic blocking agents, but without this
property, shares with Dibenzyline certain meta-
bolic actions on hepatic ferritin systems (Baez
et al. Federation Proc., this issue) consisting in
inhibition of anaerobic release of ferritin and
preservation of ferritin-inactivating mechanism
against anaerobic deterioration. The present
study reports effects of G-D 131 on mortality in
rats from standardized hemorrhagic shock with
self-infusion reservoir, when given after 90 min.
of hypotension (blood loss 2.8-3.0%), and followed
by 23 hr. drastic hypotension (30 mm Hg). Dosage
of G-D 131 was 80 ug/100 gm body weight i.v. in
0.2 cc saline. Controls received saline or Diben-
zyline, 5-20 ug/100 gm. G-D 131 more than doubled
control survival rate; Dibenzyline was actually
deleterious in contast to its pretreatment protec-
tion. It is suggested that beneficial action of G-D
131 is related to its above mentioned metabolic
effects on ferritin systems which are unaccom-
panied by undesirable side effects such as those
exerted by Dibenzyline. The status of hepatic
ferritin systems at the end of shock syndrome
and the accompanying peripheral vascular pat-
terns as influenced by G-D 131 will be discussed in
relation to protection observed.

577. Effect of independent alterations of
stroke volume, aortic pressure and heart
rate on left ventricular function. W. N.
SrainsBy, 8. J. Sarnorr, E. Braunwa tp,
R. B. Casg anp G. H. Wetca (introduced by
J. A. SHannon). Lab. of Cardiovascular Hemo-
dynamics, Natl. Heart Inst., Bethesda, Md.

An experimental canine preparation, with a
complete circulation, was devised (BRAUNWALD
et al. Federation Proc. this volume) in which
stroke volume, mean aortic pressure and heart
rate could each be held constant or varied inde-
pendently over wide ranges. The effect was ob-
served of altering each of these hemodynamic
parameters on left ventricular function (i.e.
relationship between filling pressure and stroke
work). At a constant heart rate, by progressively
increasing the stroke volume, several ventricular

1 Deceased.

178

function curves were obtained, each at a different
aortic pressure. There was an increase in the
height of each ventricular function curve as the
mean aortic pressure at which it was obtained
was elevated from 40-150 mm Hg. Above this gen-
eral level, however, the height of the function
curve decreased. Starting at low stroke work
levels, higher work levels were achieved by elevat-
ing either aortic pressure or stroke volume. It
was noted that higher filling pressures were re-
quired when work was increased by increasing
stroke volume than was the case when identical
stroke work levels were obtained by raising aortic
pressure. Function curves were also obtained at
heart rates between 60-200/min., each at a con-
stant aortic pressure, by increasing stroke volume.
The highest function curves were obtained at the
lowest heart rates and the curves became pro-
gressively lower as the heart rate at which it was
obtained was elevated.

578. Effects of constriction of carotid arteries
on renal exchanges of water, sodium and
total solutes in unanesthetized, Ringer’s
infused dogs. J. STAMLER AND L. DreErFus.*
Cardiovascular Dept., Med. Research Inst.,
Michael Reese Hosp., Chicago, Ill.

Previous work in this department led to the
conclusion that altered renal sodium-water
handling in edema-forming states occurs in re-
sponse to changes in total organism fluid
homeostasis. Function of the kidneys changes
under the influence of multiple receptor-effector
ares, nervous and humero-hormonal, arising
extrarenally. The present study was undertaken
to investigate one possible receptor-effector
mechanism, by analyzing the effects of carotid
artery constriction. Dogs previously prepared
with Van Leersum loops (skin tunnels exterioriz-
ing the common carotid arteries) were loaded
with Ringer’s solution (9-10 cc/min. i.v.) until a
constafit urine flow was achieved. After 3 control
periods, both carotid loops were constricted until
only a faint pulsation was palpable distally. Three
experimental clearance periods were then com-
pleted. Constriction engendered an immediate
significant rise in renal plasma flow (RPF), with
little or no change in glomerular filtration rate
(GFR). Concomitantly, urine flow, sodium and
total solute excretion increased; percentage ex-
cretion of filtered H,O, Na and total solutes also
increased. Following release of the constriction,
all clearance values immediately returned to con-
trol levels. Preliminary data suggest that renal
denervated dogs responded similarly to carotid
artery constriction. These results demonstrate
that renal water-sodium-total solute excretion
respond to changes in the circulation through the
carotid arteries, mainly by alterations in renal
tubular function.

FEDERATION PROCEEDINGS

Volume

579. Insulin-induced inhibition of estrogen
anti-atherogenesis in the coronary vessel
of cholesterol-fed cockerels. J. STamizp
R. Pick anp L. N. Katz. Cardiovascular Dept,
Med. Research Inst., Michael Reese Hosp,
Chicago, Ill.

Previous work demonstrated that estrogens
prophylactically inhibited coronary atherogenesis
in cholesterol-fed cockerels. This estrogen anti-
atherogenesis was effective in birds concomitantly
treated with androgens, or pancreatectomized, or
rendered hyperadrenocorticoid and diabetic by
hydrocortisone administration. It was sig.
nificantly reduced by hypothyroidism. The
present study assessed the effects of insulin on
estrogen anti-atherogenesis in intact cholesterol-
fed cockerels. Four groups of chicks, fed mash
supplemented with 2% cholesterol plus 5% cotton-
seed oil, concomitantly received the following
hormone treatments during 14-21 weeks of age:
group 1, none; group 2, estrogens; group 8, insulin;
group 4, estrogens plus insulin. Both insulin.
treated groups exhibited significant hypoglycemia
as well as reduced feed intake, growth, and de-
velopment. The three hormone-treated groups
had similar levels of hypercholesterolemia. Both
estrogen-treated groups exhibited the _ usual
estrogen-induced marked hyperphospholipemia,
with reduction of (cholesterol) /(lipid phosphorus)
ratios toward normal levels; however, this was
significantly less pronounced in group 4. In con-
trast to the estrogen-treated cockerels (group 2),
exhibiting the usual freedom from coronary
lesions, the estrogen plus insulin-treated birds
showed a significant incidence of coronary athero-
sclerotic lesions. It is concluded that insulin, like
hypothyroidism, inhibits the ability of estrogens
to protect the coronary vessels of cockerels against
cholesterol-induced atherosclerosis. These findings
pose the problem of the possible role of insulin
therapy in depriving middle-aged diabetic women
of the natural protection of their sex against
coronary disease.

580. Electrical studies of reflex patterns in
intact animals. LAWRENCE STARK AND
GriLBeRT H. GLAsER (introduced by Joun F.
Fuutron). Section of Neurology, Yale Univ.
School of Medicine, New Haven, Conn.

This investigation represents the development
of a technique of implanting permanent stainless
steel electrodes within polyethylene cuffs about
nerves and muscles. Classical neurophysiological
studies on reflex patterns were performed in the
past on central nervous systems grossly modified
surgically or pharmacologically. The present
electrophysiological data have been obtained
in awake, neurologically intact animals (cats)
and can be compared directly with the former
studies. Initial observations have shown the gross

vol

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March 1956

modifications of reflex patterns induced by com-
monly used anesthetic agents such as barbiturates.

581. Relation of peripheral pulse pressure to
eardiac stroke work and stroke volume.
Isaac Srarr. Dept. of Therapeutic Research,
Univ. of Pennsylvania, Philadelphia.

In 1928, Liljestrand and Zander proposed the
following relationship: cardiac stroke volume =
K(pulse pressure + mean pressure). In 1940, Aperia
pointed out that, since cardiac stroke work = stroke
volume X mean pressure, then by substitution,
cardiac stroke work = K(pulse pressure). This
conception has been tested by our data from 51
simulated systoles secured in 6 subjects at ne-
cropsy. From continuous curves of output and
aortic arch pressure, stroke work was estimated by
integration by Dr. Walter Feder. A comparison
has been made of these accurate estimates of work
with the corresponding peripheral pulse pres-
sures. Correlations between stroke work and pulse
pressure were found to be of a high order in each
subject; somewhat less for the group as a whole
because K varies from subject to subject, having
arelation to the subject’s age. For all 51 estimates,
the correlation coefficient between cardiac stroke
work and peripheral pulse pressure is 0.84; while
in the same data, the relation between cardiac
stroke volume and peripheral pulse pressure is
only 0.40. Thus, pulse pressure is much more
closely related to the heart’s work than to its
output. When cardiac stroke work is related to
peripheral pulse pressure + age, the correlation
coefficient rises to 0.89. Obviously there is a very
close relation between pulse pressure and the
heart’s work, and the viewpoint of the Scandi-
navian school is strongly supported by our results.

382. Elimination of carbon'-bicarbonate
following its introduction into the stomach
of the rat. F. R. SteGGERDA AND D. R. ASHER.*
Dept. of Physiology, Univ. of Illinois, Urbana,
and Dept. of Pharmacology, Abbott Labs., North
Chicago, Ill.

Earlier investigations showed that when 300-500
cc of COz is introduced into the stomach of man,
considerable amounts may be absorbed and ex-
creted via the lungs. To further test this observa-
tion, rats were given by stomach tube 3 levels of
C“ labeled sodium bicarbonate in doses of 100,
00 and 500 mg/kg. The CO» expired was collected
in chains of absorbing bottles and then concen-
trated and counted to determine the percentage
expired as C''Oo. A total of 11 different samples
were collected over a period of 24 hr. The range of
lime for each collected sample varied from 15
nin. for the 1st sample to 12 hr. for the last. The
presence of C'* in the excreta was also determined
by washing out the animal container at the end
of the experiment. The results show that over
10% of the C'4 was accounted for in the expired air

AMERICAN PHYSIOLOGICAL SOCIETY

179

in less than 2 hr. and that more than 90% is con-
sistently recovered via the expired air in the 24
hr. experimental period. The amount of C™ re-
covered in the excreta is usually less than 1%.
The results also suggest that the lower the dose the
faster the recovery of the C' in the expired air.
In experiments in which dilute HCl was introduced
into the stomach immediately after the NaHCO;
the presence of C'4O. in expired air appeared at a
faster rate than without acid ingestion.

583. Reconciliation of conflicting reports and
further data on blood volume in can-
cer. J. L. STEINFELD (introduced by H. W.
CHALKLEY). Natl. Cancer Inst., Bethesda, Md.
Blood volume determinations using the isotope

dilution technique with I'*' albumin or sodium

chromate®! homologously labeled red blood cells
have revealed an oligocythemia along with a nor-
mal plasma volume in over 60 patients with far
advanced carcinomas. Blood volume and red cell
mass were calculated utilizing the concept of

‘body hematocrit,’ with the ratio of body hemato-

crit to the peripheral venous hematocrit being

0.91. This concept permits accurate estimation of

both red cell mass and plasma volume, using

either a red cell or plasma label. Simultaneous or
serial estimations of blood volume, using both red
cell and plasma labels, yield identical values for
red cell mass and plasma volume, using the con-
cept of body hematocrit and, in fact, permit
calculation of the body hematocrit. In several
published reports (Kexuy et al. Cancer Research
12: 814, 1952; (Bateman. Blood 6: 639, 1951)
showing normal total red cell mass in cancer pa-
tients a plasma label was used. Introduction of
the correction factor for body hematocrit yields
values for red cell mass comparable with those to
be reported. Similarly it will be shown that a red
cell label and peripheral venous hematocrit will
underestimate plasma volume but may accurately

estimate red cell mass (BERLIN et al. Cancer 8:

796, 1955). Thus, the published contradictory

studies on blood volume will be shown to be in

harmony with each other and with the present
study through the introduction of the now well
recognized concept of ‘body hematocrit.’

584. Effects of cerebellar stimulation on post-
ictal depression in cats. Peter A. STEWART
AND Harry L. WIturaMs (introduced by JoHN
Hatpr). Div. of Basic Health Sciences, Emory
Univ., Emory University, Ga.

A maximal electroshock seizure produced in a
curarized cat by transfrontal stimulation is fol-
lowed by a period of relative electrical silence
in the EEG, and a prolonged period of slow wave
activity and spiking. This ‘postictal depression’
has been attributed to metabolic exhaustion of
the brain. Square wave stimulation of the cere-
bellum at 100-300/sec., either across the

180

cerebellum or via bipolar electrodes in the dentate
or interpositus nucleus during this period results
in immediate return of the postictal EEG to a
persistent normal waking pattern. Similar stimu-
lation of various areas of the cerebral cortex up to
seizure threshold does not produce this effect.
Specific pathways for this activation of the post-
seizure EKG are not yet known. That the reticular
activating system plays a part is suggested by
the known influence of cerebellar stimulation on
this system. Cerebellar activation of the post-
seizure EEG indicates strongly that the postictal
depression is not eneitrely due to metabolic ex-
haustion, but is the result of a more complex
process.

585. Hyperthermia and intestinal motility in
rats. J. CuirrorD STICKNEY, Davip W.
NortTuup AND Epwarp J. VAN LirReE. Dept. of
Physiology, School of Medicine, Univ. of West
Virginia, Morgantown.

Propulsive motility was determined in adult
rats by observing, after killing, the length of
small intestine traversed in 20 min. following the
gastric intubation of 2 ml of a charcoal suspension
in aqueous gum acacia solution. In 2 groups of
experimental rats, body temperature was elevated
by keeping the rats in the field of a diathermy
machine. The elevation was produced during 5
min. before intubation and was maintained until
killing for removal of the small intestine. Control
rats were handled similarly, except that the
diathermy machine was not turned on. In the
first experimental group the preintubation body
temperature averaged 40.1°C, or 1.9° above that
of the control group. No statistically significant
difference was seen in the 9 pairs of control and
experimental rats in which 61 and 538% of the small
intestine was traversed respectively. In the second
experimental group the body temperature
averaged 41.8°C, or 3.7° above that of the control
group. @he percentage of the intestine traversed
in the 8 control rats was 51 as compared with 24
in 9 experimental rats. The difference of 27% is
statistically significant at less than the 0.1% level
and is evidence that severe elevations of body
temperature depress motility in the rat.

586. Comparison of rates of red cell destruc-
tion produced by Hufnagel and elastic
silicone valves in dogs. FREDERICK STOHLMAN,
Jr., Stantey J. Sarnorr, Rosert B. Case
AND ZENA J. TayLor (introduced by RonaLp
E. Scantiesury). Natl. Insts. of Health, Be-
thesda, Md.

The rate of red cell destruction was only slightly
increased when the Hufnagel lucite ball-valve was
placed in the thoracic aorta in dogs with moderate
aortic insufficiency. However, when this valve was

FEDERATION PROCEEDINGS

Volume 15

incorporated into a prosthesis which was placed
between the left ventricular apex and thoracig
aorta in dogs with ‘aortic stenosis’ severe hemoly.
sis resulted. In 12 of 15 dogs, anemia of varying
severity (Het of 15-35), together with reticulo.
cytosis, hemoglobinemia, intermittent hemo.
globinuria and _ hemosiderinuria, persisted
throughout the period of study (up to 1 yr.). Red
cell survival was reduced in the 6 dogs studied. In
these dogs, in which the hematocrit ranged from
21-48, the apparent Cr*! half time varied from
3-12 days (average = 6 days) compared to 4
normal of 21-30 days. While the Hufnagel valve is
suitable for use in the aorta, it is clear from the
above data that its destruction of red cells pre.
cludes its use in direct continuity with cardia¢
chambers. With this in view, a ball-valve with an
elastic silicone housing was designed. The resilient
seat of this valve diminishes the impact force of
the ball and consequently the rate of red cell
destruction. Observations on plasma hemoglobins,
hematocrits, reticulocyte levels, and red cell sur-
vival together with the absence of hemoglobinuria
indicate that this valve is suitable for use between
the left ventricle and aorta.

587. Relation of thermal pain and tissue
injury to stimulus intensity-time and skin
temperature. ALice M. SToLt anp JAMgs D,
Harpy. Aviation Med. Acceleration Lab., J ohns-
ville, and Dept. of Physiology, Univ. of Penn-
sylvania Med. School, Philadelphia, Pa.

In the study of the relationship between pain
and tissue injury produced by thermal radiation,
direct measurements were made of the skin tem-
perature before, during and after exposure to
various irradiances for measured times. From
these data the strength-duration relationship for
threshold burn production was determined and
found to be analogous to that previously deter-
mined for threshold pain. When the intensity of
stimulus productive of the endpoint was related
to the reciprocal of time, each set of data yielded
a straight line different from the other in slope.
On extrapolation, both lines intercepted the
ordinate at the same point indicating that radia-
tion of an intensity of about 50 mc/cm?/sec. is
the threshold irradiance for both pain and tissue
damage. Injury produced during burning was
evaluated in terms of the integral of the rate of
protein inactivation at the temperature of the
skin during irradiation, computed after the
method of Hendriques and Moritz. Values thus
obtained were approximately 0.1 as large as those
obtained for comparable burns by these investiga-
tors. The discrepancy may be due to the difference
in method of heat application indicating that
formulations of data appropriate to prediction of
injury from elevated skin temperature maintained

lume 16

placed
horacie
1emoly.
varying
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hemo.
arsisted
*.). Red
lied. In
2d from
d from
d toa
valve is
‘om the
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id skin
MEs D.
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en pain
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. From
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nsity of
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ng was
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March 1956

at constant levels are not strictly applicable to
situations in which skin temperature changes
continuously throughout the thermal exposure.

588. Carbon sources for ergosterol and fatty
acid synthesis in Neurospora crassa. DaviID
Strong, OscaR HEcHTER AND ALEX NussBAuM.*
Worcester Fndn. for Exptl. Biology, Shrews-
bury, Mass.

The biosynthesis of ergosterol has been investi-
gated in an acetateless strain (No. Sy-Ac-3-Sp)
of Neurospora crassa. This mutant cannot grow
in the minimal medium of Beadle (consisting of
sucrose, ammonium tartrate, salts and biotin)
in the absence of added acetate, presumably be-
cause it cannot convert sucrose to C2 units at a
significant rate. When the mutant is cultivated in
the presence of acetate-1-C™ for 84 hr., it is found
that ergosterol and the fatty acids (FA) formed
during growth have equivalent specific activities
(SA). However, the SA of these products is only
4 to 3 of the SA of the acetate used as substrate,
indicating a carbon source for lipid synthesis
other than exogenous acetate. Comparative
studies with the wild strain (No. 74A) and the
acetateless mutant reveal that the unknown pre-
cursor a) is utilized to a greater extent than is
exogenous acetate for lipid synthesis; 6b) acts
throughout the culture period; and c) contributes
to lipid synthesis at a rate dependent upon the
amount of acetate available for metabolism. To
differentiate between sucrose and tartrate as the
carbon source, the mutant was grown in a medium
containing sucrose and acetate-1-C'* but no
tartrate. Under these conditions the ergosterol and
FA synthesized had a SA approximately equiva-
lent to that of the acetate used. These data sug-
gest that tartrate, while incapable of supporting
growth of the mutant in the absence of exogenous
acetate, nevertheless can serve as a carbon source
for sterol and FA synthesis in the presence of
metabolizable acetate. Preliminary evidence
indicates that addition of butyrate to the medium
can counteract the ‘tartrate-dilution’ effect.
(Supported by The Commonwealth Fund.)

589. Mechanisms of fluid accumulation in
ascites tumor growth. Rosert L. StravuBe,*
Marian S. Hiti* ano Harvey M. Parr. Div.
of Biological and Med. Research, Argonne Natl.
Lab., Lemont, Til.

During the early growth phase of many ascites
tumors a linear relationship exists between the
number of tumor cells and the volume of ascitic
fluid. The presence of Ehrlich ascites tumor cells
in the peritoneal cavity of mice leads to an in-
creased accumulation there of I'*!-labeled protein
after its intravenous injection. The amount of
protein accumulated is a direct function of the

y was tae

AMERICAN PHYSIOLOGICAL SOCIETY

181

number of tumor cells inoculated over a given
range. This is a local phenomenon and can be
demonstrated within 30 min. after tumor cell
injection. Generalized physiological concepts of
body fluid regulation may be applied. Although
these observations suggest an alteration in vascu-
lar permeability, it is believed that interference
with removal mechanisms plays an important
part, for the following reasons: 1) there is a sig-
nificant increase in retention of labeled protein
after intraperitoneal injection in the presence of
ascites tumor cells; 2) protein concentration of
ascitic plasma and normal peritoneal fluid is
similar (2.6 gm %); 8) blocking of lymphatics
with carbon particles increases the intraperitoneal
accumulation of intravenously injected labeled
protein in normal but not in tumor bearing mice.
The mechanisms by which the decreased peri-
toneal outflow is mediated are not obvious, but
it is believed that mechanical blockade of
lymphatics by tumor cells is not involved since
90-95% of the injected cells can be recovered after
an increased protein accumulation 1 hr. after
inoculation. The immediacy of the response and
the previously described exponential growth with
little or no lag phase also support this conclusion.
(Work performed under the auspices of the U. S.
Atomic Energy Commission.)

590. Variations of the plasma carbonic acid
pK’ with pH and temperature. M. Sruprst,
J. W. Severrnenaus anv A. F. Brapuey (in-
troduced by R. P. Akers). Natl. Heart Inst.,
Bethesda, Md.

The px’ of carbonic acid in serum and plasma
has ordinarily been assumed independent of pn.
For many years the accepted value of 6.10 at 38°
has been used to relate pCO2, CO» content and pu
according to the Henderson Hasselbalch equation.
Physiologic studies of the effects of hypothermia
on blood gases led to a reinvestigation of the
effects on pK’ of variations in temperature and
pH. The px’ was measured 39 times on serum or
plasma from 7 healthy men and 2 dogs after sam-
ples had been equilibrated in a tonometer with
known CO: tensions at 37.5° and 24°C, pH was
measured at tonometer temperature, and was
shown to have anexpected accuracy of +0.01 units,
including the National Bureau of Standard’s
uncertainty of +0.005 units. CO: content was
determined manometrically. CO. solubility was
corrected for temperature. Data from the litera-
ture and our data agree in suggesting for man at
37.5° and pH 7.4 a px’ of 6.09. The px’ was found
to rise 0.044 units for a pu fall of 1.0 unit at 38°;
at 24° this rise was 0.063 units, px’ rose 0.0054
units per degree fall in temperature at px 7.4;
at pH 6.8 this rise was 0.0063 per degree. A nomo-
gram has been constructed allowing the selection

182

of the appropriate pk’ at any particular pH and
temperature in the ranges from 6.6 to 8.0 px, and
10°-45°C.

591. Inheritance of blood pressure and its
relationship to mortality in chickens. PauL
D. Strurxiz, R. K. Rincer* anv H. 8. Werss.*
Rutgers Univ., New Brunswick, N. J.

Systolic blood pressure was determined on White
Leghorn chickens at 7-10 mo. of age in 1950, 1952
and 1954. Records of mortality and egg production
were kept until the birds were 19 mo. of age, or
until the completion of the first laying year. The
birds were divided into 3 groups with respect to
blood pressure: high, median and low. The per-
centages of birds in each category for the 3 years,
based upon 317 females and 158 males, were 28.2,
42.5 and 29.2 respectively. The death losses for
each group during a period of 9 to 12 mo. were:
high, 14.3; median, 14.8 and low, 32.5%. Egg pro-
duction was not significantly different among
the groups. Hypotensive and hypertensive strains
were developed by breeding. Males and females
having high and low pressures were selected and
the appropriate matings were made. This was done
on a small scale with only 3 breeding pens for each
line. Blood pressures were determined on the
first generation progeny at 7-10 mo. of age. The
mean differences in the pressures of the high and
low lines were 16.0 mm Hg for the male and 13.7
for the female progeny. This significant difference
indicates that the degree of inheritance (herit-
ability) of blood pressure is relatively high.

592. Effects of cooling on central nervous
system responses. Isamu Supa,* Kiyomi
Koizumi* AND CHANDLER McC. Brooks. Dept.
of Physiology, State Univ. of New York, College
of Medicine at New York City, Brooklyn.

In previous studies (J. Neurophysiol. 18: 205,
1955) it was found that cooling the cord by lower-
ing thé temperature of the surrounding medium
caused marked augmentation of spinal reflex
responses before depression occurred. It was
thought that temperature gradients within the
cord might be partly responsible for these aug-
mented responses. It was found, however, that
when the spinal cord was cooled by cooling both
the surrounding medium and aortic blood supply-
ing the cord, in order to keep the temperature
gradient of the cord minimum, reflex responses
were likewise augmented between certain critical
temperature ranges (35-27°C). Similar studies
were made of the effects of cooling on the electro-
corticograms and evoked potentials recorded
from the sensory-motor cortex and from the cere-
bellum. Cooling of the blood reaching the brain
and cooling of the brain surface alone or in con-
junction with blood cooling also produced a phase

FEDERATION PROCEEDINGS

Volume 16

of augmented response within a temperature range
of 34°-24°C. During this period the negative
phase of the evoked potentials was augmented and
the duration was much prolonged. The changes
occurring in evoked potentials recorded from the
cerebellum suggest that there is an augmentation
of cellular discharge. ECG records show that
there was an increase in amplitude between
37°-24°C, though the wave frequency remained
the same. Below this temperature range depression
predominated in all recordings and desynchroniza-
tion of evoked responses occurred. (Supported by
a grant (B-847) from the Natl. Inst. of Neurologi-
cal Diseases and Blindness, PHS.)

593. Determination of carnosine and an-
serine. D. C. Sutrin,* H. M. Hines anp T. C,
Winnick. Depts. of Physiology and Biochemistry,
State Univ. of Iowa, Iowa City.

A procedure was developed for the chromato-
graphic separation of carnosine and anserine
from each other and from the basic amino acids,
This was accomplished by a modification of the
procedure of Moore and Stein using 0.9 x 50 cm
columns of a sulfonated polystyrene cation ex-
change resin (Dowex 50-x4) as the stationary
phase and sodium acetate-citrate buffers of
gradually increasing pH and ionic strength as the
mobile phase. The recoveries of u-histidine, pL-1-
methylhistidine, L-carnosine, and DL-anserine
were approximately 90% of theory. By compari-
son, lysine was quantitatively recovered.

This procedure has been applied to the deter-
mination of carnosine and anserine in protein-
free perchloric acid extracts of cat gastrocnemius
muscle. The identities of the peptide peaks of the
resulting chromatogram were ascertained by a)
demonstrating the presence of beta-alanine and
l-methylhistidine in a hydrolysate of the
anserine peak and beta-alanine and histidine in
a hydrolysate of the carnosine peak, and b) effect-
ing an increase in the size of the respective peaks
by the addition of synthetic carnosine and
anserine to an aliquot of the musch extract before
chromatography.

594. Changes in the blood clotting mecha-
nism of cold adapted rabbits. G Bonar
SUTHERLAND (introduced by A. vaN Harre-
VALD). Gates and Crellin Labs. of Chemistry,
California Inst. of Technology, Pasadena.
Although changes in the clotting mechanism are

well established in hibernation and estivation,

only sporadic and largely unconfirmed reports
have appeared regarding changes as a result of
cold adaption. The present report concerns studies
made in this laboratory which have demonstrated
that alterations occur in the clotting mechanism
of healthy, shaved rabbits after exposure at 4°C

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March 1956

for 2 mo. This effect on the clotting mechanism
was shown by a 30-40% increase in whole blood
and plasma clotting times, and by a 35% decrease
in prothrombin time. An apparent hemoconcentra-
tion was indicated by increases in platelet (14%)
and erythrocyte (6%) counts, hematocrit (7%),
and total plasma protein (4%). There was also a
decrease in albumin (138%) and an increase in
s-globulin (37%) and fibrinogen (55%) as deter-
mined by electrophoresis. No residual fibrinogen
was present in the serums from either group of
animals. It is concluded that the increase in blood
and plasma clotting times are probably the result
of a defect in the thromboplastic mechanism,
perhaps because of increased platelet resistance.
The decrease in prothrombin time could be at-
tributable to a number of factors, but may
possibly be explained by the increased fibrinogen
(and fibrin yield) from the cold adapted animals.

59. Relations between a known volume pulse
and resulting pressure pulse. James H. R.
SUTHERLAND (introduced by W. F. Hamriton).
Depts. of Physiology and Pharmacology, Med.
College of Georgia, Augusta.

A pump has been designed with certain char-
acteristics which can be varied and quantitatively
controlled. These are rate, stroke volume, ejection
contour, and relative duration of ejection as com-
pared to the total cycle time. It consists of a
piston loosely coupled to a conventional Dale-
Schuster lever system. Rigid hydraulic trans-
mission from the piston to a finger cot actuates a
secondary pump that draws blood from the left
atrium and a flexible rubber reservoir and delivers
it through a large cannula in the brachiocephalic
artery into the aorta. When the aorta is occluded
by a heavy ligature proximal to the brachio-
cephalic artery, coronary flow and_ right
ventricular functions are preserved, and the pump
replaces the left ventricle in maintaining systemic
pressure. The pump gives pressure pulsations simi-
lar to the natural pulses, These experimentally
produced pulses will be analyzed quantitatively
under various physiological conditions in the dog.
Experiments on elastic models will also be pre-
sented. (From a Cardiovascular Research Training
Program supported by grants from the PHS and
the American Heart Assoc.)

9%. Nature of fluids in functionally dis-
tended kidney. H. G. Swann anp A, A.
Ormssy.* Depts. of Physiology and Biochemistry,
Univ. of Texas Med. School, Galveston.

When the functioning kidney is allowed to drain
ifter clamping its artery, there flows out of the
vein a fluid which has about half the hematocrit
of systemic blood. Analyses of this fluid were made
ind of systemic arterial blood, renal venous blood

AMERICAN PHYSIOLOGICAL SOCIETY

183

and urine. It was found that the fluid draining is
a mixture of vascular blood and another fluid.
The latter is designated as ‘diluting fluid.’ Its
composition was deduced from the measured
compositions of renal venous blood and of fluid
draining from the kidney after circulatory arrest.
It is not related to urine. Its content in various
substances shows the following ratios to the con-
tent of the same substances in renal venous blood
drawn shortly before arterial clamping: Na, 1.0;
Ca, 1.0; K, 1.5; Cl, 1.2; PO,, 1.8; urea, 1.8; protein,
.38; glucose, .4; inulin, .5; Diodrast, 1.9; and osmo-
larity, 1.2. Because kidney lymph is known to
have essentially the same content in 4 of these
substances (protein, urea, glucose and inulin), it
is concluded that the ‘diluting fluid’ is very prob-
ably renal interstitial fluid. This reenters the
vascular tree under the conditions of the experi-
ment. The volume of interstitial fluid is large:
some 13% of the functional renal volume and,
roughly, 20 times greater than capillary volume.
Its natural site is suggested to be, perhaps in
large part, in the extravascular ‘cisterns’ recently
discovered in the functional kidney by Pease
(J. Histochem. & Cytochem. 3: 295, 1955).

597. Effect of temperature on interrelations
between thyroxin and adrenaline. Herp1 E.
Swanson (introduced by H. N. Harxrns).
Dept. of Physiology and Biophysics, Univ. of
Washington School of Medicine, Seattle.

The influence of thyroxin on the calorigenic re-
sponse to adrenaline was calibrated by measuring
the oxygen consumption and body temperature
of thyroidectomized rats receiving daily doses of
0, 6, 12, 24 and 48 ug of thyroxin before and after
adrenaline injection, and living at 30°C, 18°C and
10°C. The calorigenic action of adrenaline was
related to the thyroxin level. The increased metab-
olism with decreased temperature may be partly
chemical and partly muscular. In cold adaptation,
an increase in the chemical component may spare
the muscles. This may be due to thyroxin-
adrenaline interrelationships. In comparison with
metabolic rates consequent to administered thy-
roxin, the oxygen consumption of intact rats,
measured at 30°C, indicated a doubling of thyroxin
secretion after 2-5 weeks’ exposure to 5°C, and
the response to injected adrenaline was corre-
spondingly greater. Assuming that endogenous
adrenaline secretion is maximal during cold ex-
posure (as suggested by the refractility to exoge-
nous adrenaline when oxygen consumption is
measured at 10°C), then the function of increased
thyroxin secretion in cold-adapted animals is to
increase the effectiveness of endogenous adre-
aline. The response to administered adrenaline at
30°C confirms the increase in thyroxin activity.
(Supported in part by the USAF under Contract

184

No. AF 18(600)-1467, monitored by the Alaskan
Air Command, Arctic Aeromed. Lab.)

598. Immediate effect of X-rays on the DPN-
DPNH relationship in grasshopper eggs.
THEODORE N. TAHMISIAN AND BeEtty JEAN
Wricut.* Argonne Natl. Lab., Lemont, Il.
Grasshopper eggs in the diapause stage of de-

velopment were used to determine whether the

DPN-DPNH balance in vivo is changed under

irradiation. Theoretically, the radicals formed

from H:0 and O: by radiation have a half life of

1 X 10° sec. to 1 X 107 sec. Therefore at any

dose rate, the rate of disappearance of radicals

should equal the rate of formation within 1 usec.,
and the resulting constant leve) attained should be
dependent on dose rate rather than on accumu-
lated dose. The grasshopper eggs, which are
approximately 5 mm in volume, were introduced
into boiling water while under the x-ray beam, to
fix the DPN-DPNH relationship during the period
of irradiation. Controls consisted of eggs treated
in the same manner except that they were not
irradiated. A 3rd group of eggs was irradiated and
then killed, to determine whether partial recovery
toward the DPN-DPNH relationship of the con-
trols would occur. In eggs killed while under
x-ray beam the ratio of DPN to DPNH content
is higher than that of the controls. In eggs killed

a few minutes following irradiation, this ratio of

DPN to DPNH is higher than that of the controls

but lower than that of eggs killed under the beam.

This suggests partial reduction following irradia-

tion. On the basis of the above observations we

may assume that when biological material is
irradiated in the presence of oxygen more oxida-
tive than reducing radicals are formed. (Work
performed under the auspices of the U.S. Atomic
Energy Commission.)

599. Early (3-5 day) mortality in rats given
suprglethal whole body x-irradiation and
the protective effect of fasting. 8. T. TAKETA
AND M. N. Swirr (introduced by Frank W.
Weyrmoutn). U. S. Navat Radiologicat Defense
Lab., San Francisco, Calif.

Mortality resulting from a supralethal whole
body dose of x-radiation (825 r) occurs predomi-
nantly during the 3rd—5th days following exposure,
and is associated with intestinal injury. Deaths
during this period are reduced from approximately
75% to 30% by fasting the animals for 24 hr. prior
to irradiation. This protective effect is not altered
by deprivation of food and/or water during the
few days subsequent to exposure. The reduction
in mortality does not appear to be correlated with
and histologically demonstrable difference in
either the degree of damage or the rate of regenera-
tion of intestinal epithelium. Diarrhea is

FEDERATION PROCEEDINGS

Volume 15

prominent in both fasted and fed groups from the
2nd through the 4th postirradiation days. Hemo-.
concentration, due to plasma volume loss, com-
mences on the 2nd day. By the 3rd day, hematoerit
values have risen from a normal of about 48 to
about 60. The pattern is similar for both fasted and
fed groups, except that the increment on day §
is less in the fasted animals. Hematocrits return
rapidly toward normal following day $ in animals
destined to survive the 3-5 day period, whereas
those of nonsurvivors continue to rise to values
above 65 several hours preceding death. These
data support the hypothesis that early radiation
death in the rat is attributable, at least in part,
to fluid imbalance.

600. Interrelations of CO:, O2 and vagal in.
fluences on respiratory centers. Prt-Cain
Tana AND ALLAN C. Youna. Dept. of Anatomy,
Univ. of Texas Southwestern Med. School, Dallas,
and Dept. of Physiology and Biophysics, Univ.
of Washington Med. School, Seattle.
Decerebrate cats with surgical lesions in the

pneumotaxic center were immersed in mineral oil
in a sealed cylinder. A rapid CO2 analyzer and a
flowmeter, connected to a tracheal cannula, re-
corded expired-air CO. and flow. A diaphragm
pump, producing sinusoidal changes of cylinder
volume, provided ventilation. When the pump
was stopped, spontaneous movements were im-
possible. Inspiratory and expiratory efforts were
recorded as the rise and fall in intracylinder pres-
sure, monitored with a capacitance-type manom-
eter. If pumping was stopped at the end of
expiration, apneustic breathing efforts followed,
indicating that impulses from the lungs did not
prevent inspiratory center hyperactivity. When
the pump was stopped while the lungs were dis-
tended, breathing was initially rhythmic, like
that in a cat with an intact pneumotaxic center.
Thus, the vagal impulses from statically distended
lungs sustain rhythmic breathing in the pneumo-
taxectomized cat. An increase in inspiratory
efforts with time indicated that CO2 accumulation
can overcome the effect of lung distention. Thus,
the effects of CO2 and stretch receptor action on
the inspiratory center may be inversely related.
Active expiratory efforts appeared only after
60-80 sec. of asphyxia or 35 sec. of Ne ventilation,
indicating that the expiratory center is activated
by anoxia. Since expiratory efforts appeared
earlier with the lung distended, anoxia and stretch
receptor impulses probably exert a synergistic
action on the expiratory center.

601. Influence of hemorrhage on peptidase
activity of blood and bone marrow. Myron
TANNENBAUM* AND ALBERT 8. Gorpon. Depi.
of Biology, Graduate School of Arts and Science,
New York Univ., New York City.

th
the

ume 15

om the
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atocrit
L 48 to
ed and
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rhereas
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These
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anda
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tidase
MI yRON
. Dept.
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March 1956

The rate of hydrolysis of glycylglycine (GG)
and glycylglycylglycine (GGG) by plasma, periph-
eral red cells and bone marrow of adult male rats
was examined, at different times after single and
multiple bleedings, by the micro-alkali titration
method of Grassmann and Heyde (’29). Nitrogen
analyses of the blood and marrow were made by a
micro-Kjeldahl method (Miller and Miller, °48),
and all values were expressed on a nitrogen basis.
In one group of animals, 3 ml of blood were re-
moved every other day on 3 occasions. Plasma
levels of both GG-ase and GGG-ase rose steadily,
attaining values approximately 100% greater than
normal by 4-7 days after the last bleeding, a time
associated with peak peripheral reticulocytosis.
These elevations in plasma enzyme activity oc-
curred concomitantly with significant decreases
in the 1) GG-ase and GGG-ase values of the red
cell hemolysates and 2) GG-ase activity of the
marrow homogenates. A similar decrease in the
GG-ase activity of marrow was observed in rats
subjected to a single bleeding (3-4 ml). This
decrease became more progressive, attaining levels
approximately 50% below normal by the 7th day
and returning to control values by 2 wk. after
the bleeding. GGG-ase activity of the marrow
increased initially in the singly bled group and
returned to normal by the 4th day, remaining at
this level for the subsequent 2 wk. Work is in
progress to determine whether other erythro-
poietic stimuli induce similar changes in the
peptidase patterns, of the blood and blood-forming
organs. (Supported by a grant from the National
Science Foundation.)

602. Sequelae of hypophysectomy in the
dog with particular reference to the pan-
creas. A. Taytor,* H. E. Essex anp G. A.
WRENSHALL.* Mayo Clinic and Mayo Fndn.,
Rochester, Minn., and Univ. of Toronto, Toronto,
Canada.

This report is based on 140 dogs hypophysec-
tomized by the transbuccal method. An intense
and permanent hyperemia of the pancreas, most
marked in young animals, appeared about 14
days following hypophysectomy, frequently ac-
companied at this time by hypoglycemia and
death. The extractable insulin of the pancreases
of 14 hypophysectomized animals was determined
by one of us (Wrenshall) and found to be con-
sistently lower than in 14 controls. This finding
corresponded with the histological appearance of
fewer and more faintly staining beta cell granule
in the islets of Langerhans. Cortisone, in some
animals, restored the extractable insulin of the
pancreas to control values, reduced the intensity
of the pancreatic hyperemia and helped maintain
the blood sugar level during fasting; its effect on
the islets is not yet conclusive. The effect of ACTH

AMERICAN PHYSIOLOGICAL SOCIETY

185

is also being investigated. Using alloxanized,
hypophysectomized, adrenalectomized rats an
attempt was made to determine, by biological
assay, the insulin content of the blood. The results
were equivocal but suggested that the insulin
content was slightly higher in the blood of
the hypophysectomized animal. Blood sugar levels
stabilize at lower values following hypophysec-
tomy but may fall rapidly to 20 mg % and lower
during fasting and sometimes following intrave-
nous glucose administration. The hydrocortisone
content of the blood declined to zero over a
period of about 12 weeks. This study augments the
evidence of an interrelationship of the hypoph-
ysis, adrenal gland and pancreas and may shed
some light on the etiology of diabetes mellitus.

603. Water exchange in man in the presence of
a restricted water intake and a low calorie
carbohydrate diet. HENry Lonestreet Tay-
LoR, Francisco GRANDE,* ExuswortH Bus-
KIRK,* JOSEPH T. ANDERSON AND ANCEL Keys.
Lab. of Physiological Hygiene, Univ. of Min-
nesota, Minneapolis.

The effects of water restriction in the presence
of semistarvation was studied in 12 clinically
healthy soliders aged 21 to 25 who were expending
1200 cal. in 2 hr. daily on a motor-driven tread-
mill. The men slept, ate and worked in an air-
conditioned suite at 78°F and 65% relative humid-
ity. After a control period of 21 days, all men
subsisted on 1000 cal. of pure carbohydrate. The
daily water ration was 900 cc for group I (6 men)
and 1800 cc for group II (6 men). In group I after
5 days of water restriction, there was an 8.5% loss
of body weight, a 47% reduction in the rate of
sweating during a treadmill walk of 1 hr., a 60%
reduction of water loss during 8 hr. of sleep and
an average daily urine volume of 350 cc. Water
balance calculated from weight loss, difference
between excreted and ingested solids and the
weight of fuel burned showed a water loss of 1.61.
during the Ist day and 0.4 1. during the 5th day.
Group IT, during a 10-day restriction of water,
showed smaller but still significant decreases in
sweating rates and appeared to be in water balance
between the 4th and 10th day. It is concluded
that substantial water conservation can be
achieved by men on restricted water and calories.

604. Electromyographic study of abdominal
reflexes in man. R. D. TEAsDALL* AND J. W.
Maauapery. Subdepartment of Neurological
Medicine, Johns Hopkins and Baltimore City
Hosps., Baltimore, Md.

In 14 normal right-handed individuals with
symmetrical abdominal reflexes there is consider-
able variation in latency of the reflex muscle
action potentials recorded from the surface of the

186

abdomen following cutaneous stimulation. Ab-
dominal reflexes with latencies as short as 32-48
msec. are consistently recorded in the younger
subjects, whereas in older individuals they are
considerably longer (96 msec.). There is no ap-
preciable difference in latency between the 2 sides.
In 4 individuals of this group, scratching the (L)
side of abdomen produced not only ipsilateral
potentials but also spread to the (R) side. In none
did spread occur solely in the reverse direction. In
4 subjects, however, bilateral abdominal activity
was recorded following stimulation of either side
of the abdominal wall. A similar study was per-
formed in 6 patients hemiparetic from cerebral
lesions in whom the abdominal reflexes were de-
pressed on the involved side. In these cases it was
noted that the latency of the muscle action po-
tentials was considerably longer on the hemi-
paretic side and with one exception, if bilateral
abdominal potentials were elicited, such occurred
only on stimulation of the hemiparetic side. It is
felt that in man descending spinal pathways act
by facilitating intraspinal mechanisms associated
with the abdominal reflex and that the basic
mechanism of the reflex is spinal. (Supported by a
grant from the National Inst. of Neurological
Disease and Blindness.)

605. Ketogenesis in mammary tissue. CHARLES
TERNER (introduced by GreEGory PrNcus).
N.I.R.D., Univ. of Reading, England, and Wor-
cester Fndn. for Exptl. Biology, Shrewsbury,
Mass.

Lactating guinea pig mammary homogenates
were incubated with radioactive acetate, pyruvate
or glucose in the absence or presence of fumarate.
When the fumarate concentration was high (0.005
M), substrate-C'* was predominantly incorporated
into fatty acids. When fumarate was absent or
present in low concentration, acetoacetate was the
main product. As the concentration of fumarate
was raiséd, the rate of acetoacetate formation de-
clined and the rate of lipogenesis increased pro-
gressively. Ketogenesis occurred most readily from
pyruvate, followed by acetate and relatively
slowly from glucose, even when hexokinase was
added. The oxidation of unlabeled glucose simul-
taneously with C'-acetate only slightly depressed
the radioactivity of the acetoacetate formed and
sometimes even caused stimulation of ketogenesis,
while unlabeled octanoate caused considerable
isotope dilution. p-Nitrophenol (2 X 10-4 m) or
fluoride (0.005 m) inhibited ketogenesis from ace-
tate with inhibition of O2. uptake and C™O: pro-
duction, but allowed the conversion of pyruvate to
acetoacetate to continue at almost undiminished
rates; arsenate was inhibitory. In homogenates
glycolyzing under anaerobic conditions, aceto-
acetate formation from C'-acetate was inhibited

FEDERATION PROCEEDINGS

Volume 1§

by oxaloacetate which at the same time promoted
anaerobic lipogenesis. These findings show that
liver can no longer be regarded as the exclusive
site of ketone body formation. The observed strong
ketogenic tendency in mammary tissue, antago-
nized in the present experiments only by ex-
ceptionally high concentrations of the Krebs cycle
catalyst, may have some bearing on the problem
of ketosis in lactating dairy animals.

606. Facilitation and depression of excita-
bility in artificial synapse. CaRLo A. Trr-
ZUOLO AND SvEN G. E.tasson (introduced by
JANE E. Hype). Dept. of Anatomy, Univ. of
California at Los Angeles.

Transmission across the artificial synapse be-
tween adjacent cut fibers at the proximal end of
the cat’s severed sciatic nerve (Granit et al.) was
studied. A conditioning stimulus was applied toa
dorsal or ventral spinal root contributing to the
sciatic nerve, and we recorded from the other root,
A test stimulus was applied directly to the cut
end of the nerve. The intensity of the conditioning
shock was arranged so that only a small response
would be observed in the efferent fibers from the
artificial synapse. A submaximal test stimulus was
employed. Depending on the time interval be-
tween the two shocks, pronounced facilitation or
depression of the response to the test stimulus was
observed. The facilitation was confined to a few
msec. (3-5), while the depression lasted up to 45
msec. when the conditioning shock was applied to
a ventral root. A different time course of depres-
sion was observed when the conditioning stimulus
was applied to a dorsal root. Post-tetanic po-
tentiation can occur. Since the test stimulus
was directly applied to the efferent fibers from
the artificial synapse, the excitability changes
observed must be attributed, at least in part, to
post-synaptic events. The synaptic nature of the
transmission through the cut end of the nerve is
demonstrated by its disappearance after short-cir-
cuiting the cut end with physiological saline.

607. Factors influencing phosphate Tm in the
dog. Davip D Tuompson (introduced by
JosepH C. Hinsey). Dept. of Physiology, Cornell
Univ. Med. College, New York City.
Experiments in several animal species have indi-

cated that renal phosphate reabsorption is in-

fluenced by a hormone of the parathyroid glands.

Attempts to demonstrate an effect of administered

parathyroid extract in dogs, however, have

yielded variable results. Some investigators have
found a reduction in phosphate reabsorption in
dogs upon the administration of bovine para-
thyroid extract whereas others have been unable
to establish any change. Furthermore, variability
in phosphate Tm has been noted in both dogs and

ume 1§

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March 1956

humans which has not been shown to be a conse-
quence of variations in parathyroid function.
Initial studies have been designed to assess this
variability and to evaluate the effect on phosphate
Tm of exogenous parathyroid hormone by ad-
ministering bovine parathyroid extract chroni-
cally. These results are to be compared with the
effects of high phosphate or low calcium diets
thought to pr duce an endogenous hypersecretion
of the parathyroid glands. The dietary studies
should provide information on the role of phos-
phate and calcium in the regulation of parathyroid
function. A high phosphate intake has been shown
to result in a reduction in phosphate reabsorption
in the rat and in man. This effect appears to be
mediated through an increased parathyroid hor-
mone secretion since it is not seen following para-
thyroidectomy. A change in phosphate Tm was
not seen following calcium depletion in man, but
the depletion may not have been severe enough. A
more prolonged study on the dog is being under-
taken.

608. Effect of castration and testosterone on
weight of organs and individual muscles of
the fasting guinea pig. Caro, TILLOTSON*
anp C. D. Kocuaxktan. Oklahoma Med. Research
Fndn., Oklahoma City.

Male guinea pigs of the inbred Hartley strain
were divided into 5 groups of 6 each at 500 (486-
530) gm b. wt. and 3 of the groups were castrated.
After 42 days food was removed from a normal
and 2 castrated groups. The guinea pigs of one of
the castrated groups were implanted subcutane-
ously with a pellet of testosterone (approx. 15 mg).
Nine days later the animals were killed. The 9
days without food produced an average decrease
in body weight of approximately 30% without any
difference among the 3 groups. The kidney, liver,
thymus, spleen and heart showed the expected
decreases. The seminal vesicles and prostates de-
creased 51% in the normal animals. The perirenal
fat decreased 13% in the normal and 37% in the
castrated guinea pigs. The difference between the
2 groups was not statistically significant. The
individual muscles showed an average decrease of
approximately 35% with only slight variations
among the 48 muscles. The muscles which de-
creased after castration showed only a small or no
further decrease on fasting. Testosterone was
unable to prevent the effects of fasting although it
produced a slight increase in the seminal vesicles
and prostates and the retractor penis.

609. Endocrine responses during adaptation
to moderately high altitude. Paotra S.
Trurras,* ADRIENNE A. Batts,* Grorce W.
Houiuincer,* RaupH Karwuer,* ALVIN A.
Krum* anp NELLO Pace. Dept. of Physiology,

AMERICAN PHYSIOLOGICAL SOCIETY

187

Univ. of California, Berkeley and the White

Mountain Research Station, Big Pine, Calif.

Weight and morphology of adrenals, hypoph-
ysis, pancreas, testes and thyroid were investi-
gated in rats exposed for various periods of time
at the 12,500 ft level of the White Mountain Re-
search Station (P animals), and in rats of the
second generation born at the Station (F2 animals).
Comparison was made with rats remaining in the
parent colony on the Berkeley campus (sea level
controls). Both colonies were maintained under
comparable conditions of food, caging and tem-
perature. The P animals were born at sea level and
maintained there for about 10 weeks before trans-
fer to White Mountain, 1 and 3 days and 2 months
before killing. After 1-3 days of exposure, adreno-
cortical activity was stimulated as indicated by
a) a 40-50% increase in adrenal weight, b) a loss
of adrenal ascorbic acid (after 1 day’s exposure)
and c) a 60-80% decrease in weight of thymus,
spleen and lymph nodes. No changes in weight
could be observed in hypophysis, testes and
thyroid. The preputial glands were significantly
enlarged after 3 days’ exposure. After 2 months’
exposure, the P animals showed a significant en-
largement of the hypophysis and thyroid as well
as of the adrenals even when other criteria (e.g.
growth, reproduction, blood hemoglobin and
hematocrit) indicated adaptation to the new en-
vironment. Testes and preputial glands remained
unchanged. On the other hand, in the F2 animals
born at high altitude, endocrine weights appeared
to be similar to those of sea level controls. Results
will also be reported concerning the histology of
the hypophysis, thyroid and pancreas. (Supported
by ONR.)

610. Role of thyroxin, adrenal steroids and
diet on restoration of protein and enzymes
in regenerating liver of rats. SAMUEL R.
Tipton, C. W. Masor* anv J. L. SmMoTuHeErs.*
Dept. of Zoology and Entomology, Univ. of Ten-
nessee, Knoxville.

Thyroxine treatment led to a greater restoration
of liver nitrogen and total liver succinoxidase ac-
tivity after 4 days of regeneration than did thio-
uracil. A 1% protein diet alone resulted in a signifi-
cant decrease in liver enzyme activity, but the
restoration of liver mass, protein and enzyme ac-
tivity was greater after 4 days regeneration than
in animals on 18% protein. After 7 days regenera-
tion these changes were reversed. Although the
thyroxine-treated, low-protein animals had sig-
nificantly higher enzyme activities than did the
euthyroid group, there was no significant effect of
thyroxine on the rate of restoration during the 4
days. After 7 days of liver regeneration on non-
protein diets the restoration of liver protein and
enzyme activity was significantly lowered. Previ-

188

ous adrenalectomy of rats on diets of 18% protein
resulted in a decrease at the time of liver removal
and at killing after 7 days of regeneration. Re-
ducing dietary protein to 0-2% resulted in im-
mediate crisis with death. Subcutaneous desoxy-
corticosterone and, less effectively, cortisone kept
the animals alive and led to an increase in liver
protein and enzyme during regeneration on a non-
protein diet. (Supported in part by grant A-409
from Natl. Insts. of Health, PHS.)

611. Isolations of alpha-ketolic steroids from
normal urine. JosEPH C. TouCHSTONE,* HELEN
BULASCHENKO,* Epwin M. RicHARDSON* AND
F. Curtis Donan. Endocrine Section, William
Pepper Lab. of Clin. Medicine, Univ. of Pennsyl-
vania, Philadelphia.

Previous studies on the isolation of pregnane-
3a,118,21-triol-20-one (tetrahydro B), allopreg-
nane-3a,118,21-triol-20-one (allo-tetrahydro B)
and pregnane-3a-2)-diol-11,20-dione (tetrahydro
A) from the urine of subjects treated with cortico-
tropin have been extended to the isolation of these
materials from the urine of normal untreated sub-
jects. In addition to cortisone and hydrocortisone
and their tetrahydro metabolites, corticosterone
and dehydrocorticosterone were detected in the
urine of these subjects. Following hydrolysis with
glucuronidase and acid, the steroidal metabolites
in a pool of urine of 3 normal males were extracted
with chloroform and separated by chromatography
in several different solvent systems. Initial sepa-
ration of tetrahydro B and allo-tetrahydro B was
accomplished in the toluene propylene glycol
system. Rechromatography of the eluted material
in the benzene formamide system was necessary to
separate these steroids from the large amount of
ultraviolet absorbing material present. Final
purification was achieved by rechromatography of
the acetates initially in the methylcyclohexane
propylene glycol paper chromatographic system
and then on a silica gel-Celite column. Identifica-
tion procedures in additon to other studies were
based on the following criteria: mobility in the
various solvent systems as the free steroid and as
the acetate, reaction with blue tetrazolium, nega-
tive result in the phenylhydrazine reaction of
Porter and Silber, absence or presence of absorp-
tion of ultraviolet light, absorption spectrum in
concentrated sulfuric acid and in several instances
infrared spectrometry.

612. Electrical response to sound in noctuid
moth nerves. ASHER E. TREAT* AND KENNETH
D. Rorper. City College of New York and Tufts
Univ., Medford, Mass.

Electrical responses to acoustical stimulation of
the tympanic organ at frequencies between 3 and

120 keps were recorded from the main lateral nerve

FEDERATION PROCEEDINGS

Volume 1§

trunks of the metathoracic ganglia in noctuid
moths of 5 common species. Chief among several
sound sources was a 45° x-cut crystal plate of
Rochelle salt driven directly by an electrical
oscillator and placed about 8 in. from the prepa-
ration. Moths were hemisected in the midsagittal
plane after decapitation, dealation and amputa-
tion of the legs at the coxae. The nerve was severed
from the ganglion and its free end was lifted ona
silver hook electrode. In some experiments the
alar branch of the nerve was divided, leaving the
tympanic branch intact. Responses ceased when
the tympanic branch was severed or the tympanic
organ destroyed. Most preparations showed evi-
dence of 3 active fibers, their responses character-
ized respectively by a) tall spikes appearing con-
tinuously at a rhythm more or less independent of
acoustical stimulation, and 6b) 2 shorter spikes
evoked by the sound. It is suggested that the 2
shorter spikes may be associated with the 2
scolopes typical of the noctuid tympanic organ.
Initial spike frequencies (as high as 1000/sec.)
bear no obvious relation to the vibrational fre-
quencies of the stimuli. Spike frequency drops to
25% of the initial value within 1} sec. of the stimu-
lus onset. The range of maximum sensitivity
coincides roughly with the reported range of
maximum energy emission in the ultrasonic cries
of bats.

613. Efferent impulses to the nasal area. Don
TucKER AND Lioyp M. Brrpter (introduced by
Dexter M. Easton). Dept. of Physiology,
Florida State Univ., Tallahassee.

The olfactory area of the rabbit receives un-
myelinated olfactory nerve fibers terminating in
olfactory sense cells, trigeminal nerve fibers
terminating in free endings, and fibers of the
autonomic nervous system. The electrical activity
has been recorded peripherally and centrally from
nerves identified as olfactory and trigeminal. The
peripheral activity has already been described
(Science 122: 76, 1955). On the central side the
olfactory was quiet and the trigeminal extremely
active when the rabbit was under urethane anes-
thesia. The trigeminal activity was greater and
showed marked synchronization with respiration
during deep anesthesia. The pattern of activity
was similar to that recorded peripherally from the
olfactory nerves. A pinch to the foot or a loud
noise was followed by an increase in the central
trigeminal activity. The increase was greater and
more prolonged under light anesthesia. There was
a transient quieting followed by an increase over
the initial level when odorous air was suddenly
supplied. Amyl acetate at concentrations above
olfactory threshold and below trigeminal afferent
threshold produced only the quieting effect under
proper conditions. Nearly all the central activity

je. ee

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lume 1§

noctuid
several
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mpanic
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ial fre-
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ic cries

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March 1956

in the trigeminal nerve was abolished by section
of the homolateral sympathetic nerve in the neck.
(Supported in part by a research grant from the
Armour Research Fndn.)

614. Effects of temperature on isometric ten-
sions of pellicular actomyosin fibers. BrErR-
NARD D. Tunik (introduced by Teru Hayasut).
Dept. of Zoology, Columbia Univ., New York
City.

These fibers behave mechanically as though
they were composed of a contractile, slowly re-
sponding element in series with a rapidly respond-
ing elasticity (HAYASHI AND RosenBLUTH. Proc.
Nat. Acad. Sc. 39: 1285, 1953). Experiments were
undertaken in an attempt to elucidate the nature
of the tension-bearing forces of these 2 aspects of
the fiber. The passive tension developed by stretch-
ing a fiber at 20°C was allowed to decay nearly to
equilibrium under isometric conditions. When the
temperature was lowered to 0°C the tension
changed to a new equilibrium. This change was
nearly reversible. The new equilibrium is reached
quickly (0.2 hr.) if the tension changes in the same
sense as the temperature (dS/dT is +); slowly
(0.5 hr.) if dS/dT is —. At small extensions dS/dT
is --, becoming + at larger ones. This reversal
occurs, for fibers stretched in low salt (.05 m KCl,
#s veronal buffer pH 7.6), at ca. 180% rest length
(Lo); for fibers treated with ATP (0.3% ATP and
001 m MgCl, in low salt) and then stretched in
low salt, at ca. 105% Io. Active tensions at Lo are
developed slowly by fibers to which ATP has been
added at 20°C, or by contracted fibers that have
been quick-released. Fibers in ATP at 0°C, how-
ever, develop active tensions as rapidly as the
temperature can be raised to 20°C. The interpreta-
tion of these data in terms of the contribution of
entropic and potential energy forces to the tension
will be discussed. (Supported by a Research
Fellowship of the Natl. Heart Inst., Natl. Insts. of
Health, PHS.)

615. Pulmonary carbon monoxide uptake and
diffusing capacity in normal subjects and
patients with restricted pulmonary blood
flow. G. M. Turtno,* M. BRANDFONBRENER*
AND A. P. Fisuman. Dept. of Medicine, Columbia
Univ., New York City.

The rate of uptake of carbon monoxide from
inspired gas (Vco) and the diffusing capacity of
the lung for carbon monoxide (Dco) were studied
in two groups of subjects: a) normal, b) patients
with restricted pulmonary blood flow and varying
degrees of pulmonary congestion. Pulmonary
blood flow was measured by the direct Fick
method, using the techniques of cardiac catheteri-
zation and arterial cannulation, and analyses of
blood and expired gas for oxygen content. Simul-

are tae

AMERICAN PHYSIOLOGICAL SOCIETY

189

taneous measurements of Vco and Dco were made
during a steady state, at rest, and during mild
exercise (Vo2 up to 1100 ml/min.). For the caleu-
lation of Dco, inspired gas was fixed at 0.1% CO
in air; FEco was determined by infra-red analysis,
Paco from the Bohr formula assuming Paco, =
PAcog, end expiratory Paco, by infrared analysis,
and Paco by the method of Allen and Root (J.
Biol. Chem. 214: 319, 1955). Results indicate that
at rest, during 6 min. of CO breathing, Dco calcu-
lated from Paco and Paco may exceed Deco calcu-
lated from Paco alone by up to 15%, and during 12
min. of exercise, this difference may increase up
to 30%. The Deo and Vco increased during exercise
in both groups; these increases accompanied in-
creases in minute ventilation, and occurred despite
abnormally low increments in blood flow in some
patients. In patients with pulmonary congestion
due to mitral stenosis, Dco increased normally
during exercise despite marked increases in pulmo-
nary artery pressure.

616. Purification of lipid antithromboplastin.
D. L. Turner anp M. J. Stivur (introduced by
L. M. Tocantins). Charlotte Drake Cardeza
Fndn., Jefferson Med. College, Philadelphia,
Pa.

Difficulties in dissolving the lipid antithrombo-
plastin of brain for assay (MusHEetTt, GoLDSMITH
AND Ke.uey. J. Biol. Chem. 211: 163, 1954) have
been avoided by using sodium desoxycholate in a
buffer of px 7.4 and testing in an activated plasma
system. A standard of high activity (165 Tocantins
u/mg) was used. Cyclohexane extracts of acetone-
dried brain or chloroform-methanol (2:1) ex-
tracts of fresh wet brain were treated by the
methods of Folch (J. Biol. Chem. 146: 35, 1942)
for the isolation of his fraction III, or by partitions
between cyclohexane and methanol followed by
the Folch precipitation. Material of 150-170 u/mg
thus obtained was further purified by repeated
extraction with chloroform-methanol (1:1.8) with
rejection of most soluble and least soluble ma-
terial, and also by chromatography on silicic acid
and silica gel. The chromatographic method was
effective for obtaining highly active fractions from
side-fractions like the Folch fractions I and II.
Extensive use of chromatography and solvent
partition has not resulted in enhancement of the
activity of the most active fractions obtained by
the chloroform-methanol extraction of fraction IIT
beyond 250 u/mg. Such material has phosphatidyl-
serine as its major component, but its activity does
not parallel the ninhydrin nitrogen, which may
vary from 0.4 to 1.4%. Activity is enhanced by
heating in aqueous solution or by storage. Cata-
lytic hydrogenation by the method of Baer and
Maurukas (J. Biol. Chem. 212: 39, 1955) and di-
azomethanolysis destroy activity.

190

617. Extra-hepatic conjugation of hydro-
cortisone. M. Don TuRNER (introduced by
James D. Harpy). Dept. of Surgery, Univ. of
Mississippi Med. Ctr., Jackson.

The rate of 17-hydroxycorticosterone conjuga-
tion with glucuronide has been determined in dogs
and in man by the rapid intravenous introduction
of the free steroid. Arterial blood samples were
obtained and the free and glucuronide conjugated
steroids possessing the 17,21-dihydroxy-20-keto
function were analyzed according to the methods
of Nelson and Samuels, and Bongiovanni, re-
spectively. The first-pass arterial blood samples
contained, as conjugated steroid, a large per-
centage of the injected dose. Both the free and
conjugated steroid time-concentration curves fell
exponentially and either closely paralleled or were
superimposed 2-6 min. from injection time. The
experiment suggests almost instantaneous conju-
gation and a tendency toward equilibrium between
the free and conjugated steroids, which may be
found to support the idea of the reversible nature
of conjugated steroids in the circulation. The
peripheral conjugation postulate is supported by
the finding of high concentrations of conjugated
hydrocortisone-like steroids in the adrenal veins
of human patients during surgery. Further evi-
dence for peripheral steroid conjugation has been
demonstrated by introducing the free alcohol of
hydrocortisone into freshly drawn dog plasma,
immediately extracting the free steroid and ana-
lyzing for the steroid glucuronidate by cleavage
with beta-glucuronidase. In this case the conju-
gated steroid has been found to increase from 4 to
10 times over the control values. Although periph-
eral conjugation of hydrocortisone has been
demonstrated, a functional liver would appear to
be required, since in patients who exhibit almost
nonfunctional livers conjugation is slight or un-
detectable. (Army Contract DA-49-007-MD-627.)

618. Factors affecting utilization of oxalo-
acetate (OAA) and alpha-ketoglutarate
(KG). Davip B. TYLER AND GLENN A. FiscHEr.*
Dept. of Pharmacology, School of Medicine, Univ.
of Puerto Rico, San Juan.

Using saline homogenates of rat kidney fortified
with an excess of ATP, Mg** and Versene, the
utilization of 2 keto acids was studied. OAA utili-
zation (determined directly) is inhibited by suc-
cinate when both the substrate and the inhibitor
are employed in low concentrations (0 5-2 mm).
Increasing the concentration of OAA to about 5
mM diminishes the degree of inhibition. However,
with high concentrations of OAA (10 mm), suc-
cinate stimulates rather than inhibits OAA utili-
zation. l-Malate also inhibits OAA utilization but
there is no clear lessening of the inhibition when
the concentration of OAA is increased. Fumarate

FEDERATION PROCEEDINGS

Volume 16

produces erratic inhibitory effects on OAA utili-
zation. KG utilization is also inhibited by suc-
cinate and by malate but increasing the concen-
tration of KG (up to 2 mm) does not diminish the
inhibition. Fumarate is without significant effect
on KG utilization. The total keto-acid utilization,
when a combination of both OAA and KG is used,
is much less than the sum of the individual rates
of those keto acids (as determined when they are
used alone). The results indicate that these acids
are mutually inhibitory. With the concentration
of each individual keto acid at 1 mm, the total
keto-acid utilization is 12% less than the sum of
the individual rates; when the concentration of
each keto acid is 8 mm, the total utilization is 60%
less. These data indicate that regulation of rate
through the Krebs cycle may, in part, be ac-
counted for by competiton between some of the
intermediates of the Krebs cycle. (Supported by a
grant from the Life Insurance Med. Research
Fund.)

619. Vaso-active material of guinea-pig
serum. GEORGES UNGAR AND Ricuarp £,
GoLpHAMER.* Dept. of Pharmacology, U. 8.
Vitamin Corp., Yonkers, N.Y.

Miles et al. (Brit. J. Exper. Path. 36: 71, 88,
1955) have shown that guinea-pig serum acquires
by dilution the property of producing a vascular
reaction when injected intradermally to guinea-
pigs. In the present study these observations were
confirmed and it was also found that addition of a
chelating agent (Na ethylenediamine tetraacetate)
to serum produces the same effect as dilution.
After dialysis against saline, serum loses its ac-
tivity but can be reactivated by addition of K* at
low concentrations and inactivated again by Ca**
or Mg**. These results suggest that the vaso-
active material (or its formation) requires Kt and
is inhibited by Ca** and Mg**. Preliminary data
suggest that the material may be related to the
activator of plasminogen. The relation of this
property of serum to the phenomena of ‘serotoxin’
and ‘anaphylatoxin’ formation will be discussed.

620. Additive effects of respiratory alkalosis,
carbonic anhydrase inhibition and potas-
sium on renal tubular reabsorption of bi-
carbonate. PARKER VANAMEE, FIpDEL Cwa-
sJunco, JR., Henry T. RANDALL AND KATHLEEN
E. Roserts (introduced by R. W. Rawson).
Memorial Ctr. and Cornell Univ. College of
Medicine, New York City.

It has been shown that respiratory alkalosis,
carbonic anhydrase inhibitors and potassium have
similar effects in blocking renal tubular reabsorp-
tion of base bound bicarbonate. Although it has
been postulated that these effects result from alter-
ations of intracellular pH, none of these agents

Oe ee a

ume 15

utili.
y suc-
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ation,
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l rates
ey are
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ration
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RD E.
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71, 88,
‘quires
uscular
suinea-
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on of a
retate)
lution.
its ac-
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y Cat*
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issed.

alosis,
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Cua-
‘HLEEN
WSON).
lege of

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March 1956

have been.,shown quantitatively to block bicar-
bonate reabsorption completely. Therefore, the
experiments reported here were carried out in an
attempt to produce a maximal change in the intra-
cellular pH of the renal tubules with the possibility
of determining the maximal amount of bicarbonate
transport which may be thus effected. Bicarbonate
reabsorption was determined under 3 experimental
conditions; 7) during a period of potassium
chloride or phosphate infusion which was given to
the limits of tolerance, 2) during a period of re-
spiratory alkalosis superimposed on the potassium
infusion and 3) during periods in which the animals
were given potassium, Diamox and also had a
respiratory alkalosis. The results of these experi-
ments show that potassium and a superimposed
respiratory alkalosis are additive in blocking bi-
carbonate absorption. During the periods in which
the animals were in respiratory alkalosis and also
receiving Diamox and potassium it was demon-
strated that as much as 90-98% of the filtered
bicarbonate may be excreted and reabsorption of
bicarbonate was often decreased to as little as 0.1-
0.6 mEq/100 ce of glomerular filtrate for periods up
to 60 min., despite wide variations of plasma pCO.
These studies furnish further evidence that the
intracellular pH of the renal tubules is a major
factor in governing reabsorption of filtered bi-
carbonate.

621. Demonstration of sodium in petroleum
ether extracts of skeletal muscle of the dog.
Joun C. VANATTA AND Roy M. Lanpers, JR.*
Dept. of Physiology, Univ. of Texas Southwestern
Med. School, Dallas.

Fresh muscle biopsies were taken from the
shoulder or thigh muscles of dogs under Nembutal
anesthesia. These were then weighed, dried at
110-120°C for 8-14 hr., and reweighed. The dried
material was extracted with three 10 cc portions
of dried petroleum ether leaving each portion on
the material for 8-14 hr. The petroleum ether was
washed 2 times with 10 ce distilled water, evapo-
rated to dryness, and the residue ashed with
H.SO, and HNO; at 550°C. The ash was dissolved
in water, and phosphates precipitated by adding
Ca(OH). to the aqueous solution. An aliquot was
then dried, and a sodium determination carried
out on the residue by the Butler-Tuthill technique.
This revealed some petroleum ether extractable
sodium in the residue. The amount of sodium
found was of the order of magnitude of 1.0-2.5
mEq/kg of wet muscle. Blank determinations
were carried out to check the purity of the rea-
gents used. The nature of binding to make the
sodium extractable is not known. Preliminary re-
sults indicate this sodium is labeled within 3 hr.
in vivo using Na. The suggestion is made that this
sodium may be involved in transport mechanisms

ern i

AMERICAN PHYSIOLOGICAL SOCIETY

191

across the muscle cell membrane. Preliminary
studies extracting dried plasma and red cells
indicate no comparable sodium in these tissues.
(Supported in part by the American Heart Assoc.,
and in part by research grant H-1574-C from the
Natl. Heart Inst., PHS).

622. Lactic acid analysis of post-mortem
brain tissue as aid in determination of
physiological condition prior to accidental
death. Donatp D. Van Fossan, Syrreu S.
WILKs AND RosBert T. CuLark, JR. (introduced
by Herman 8. Wicopsxy). Dept. of Physiology-
Biophysics, USAF School of Aviation Medicine,
Randolph Air Force Base, Texas.

It ‘has been demonstrated that brain lactic acid
concentration post mortem (0-6 hr.) is influenced
by status of oxygenation immediately prior to
death. This influence has been found in rat, rab-
bit and dog. Hypoxia prior to death causes an
elevated lactic acid level in brain tissue. The mag-
nitude of the rise in lactic acid has been found to
be directly proportional to both the degree and
duration of hypoxia. Although brain lactic acid
concentration post mortem may be influenced by
many other factors such as diet, hormone balance,
exercise, etc., it appears that the elevation of
lactic acid due to hypoxia is great enough to
mask the other variations. Hyperventilation, how-
ever, in the rabbit and dog produced post-mortem
(0-6 hr.) lactic acid levels as high as would be
produced by exposure to 25,000 ft. for 15 min. It is
therefore necessary to distinguish elevated lactic
acid cases due to hyperventilation from those
cases due to hypoxia. Experiments are now in
progress to devise methods of differentiating
hyperventilation cases and hypoxia cases on the
basis of a redistribution of ions in the tissues. Ef-
forts are being made to evaluate the normal limits
of lactic acid in human brain tissue at intervals up
to 24 hr. post mortem in order that lactic acid
levels may be established as a reliable post-mor-
tem test for ante-mortem hypoxia.

623. Effect of Tapazole on rat heart-weight/
body-weight ratio. Epwarp J. VAN LIERE AND
Tuomas R. Mazzocco.* Dept. of Physiology,
School of Medicine, West Virginia Univ., Morgan-
town.

Twelve female rats (Wistar strain) were given
Tapazole (1-methyl-2-mercapto-imidazole), which
is a potent antithyroid drug. The rats were given
water ad libitum containing 0.11 mg/ml for a
period of 45 days. No other water was available
for them to drink. They were fed an adequate
diet and kept at room temperature. Twelve con-
trol rats (of the same sex and strain) were kept in
an adjacent cage; they were fed the same food, but
no Tapazole had been added to their drinking

192

water. At the end of the 45-day period the animals
were weighed, stunned by a blow on the head, and
the heart removed. The large vessels were trimmed
flush with the heart muscle, the heart was washed
with tap-water, and the excess moisture removed
with blotting paper; the heart was weighed to the
nearest 5 mg. The heart-weight/body-weight
(HW/BW) ratio was expressed by the mg of heart
substance per kg body weight. The average body-
weight for the control rats was 228 gm, the average
heart-weight 725 mg and the HW/BW ratio 3.18;
for the experimental group the respective figures
were: 230 gm, 651 mg and 2.83. The HW/BW ratio
of the rats which received Tapazole was signifi-
cantly less (at the 0.1% level of confidence) than
the controls. (Supported by a grant from the
Natl. Heart Inst., PHS.)

624. Measurement of tissue gas tensions uti-
lizing gas pockets. Huau D. Van Liew (intro-
duced by J. R. Murutn). Dept. of Physiology,
Univ. of Rochester School of Medicine and Den-
tistry, Rochester, N. Y.

Artificial gas pockets were produced by sub-
cutaneous injection of air on the backs of rats.
The steady state composition which is reached
about 4 hr. after injection has been assumed to
be a measure of tissue tension by some authors. A
method of tissue tension estimation utilizing gas
pockets was devised using a series of known gas
mixtures having lower or higher O2 (or CO2) than
the steady state composition. For tissue O2 meas-
urement a given volume of one of these known
gases was injected into the gas pocket. After a
short interval the gas was withdrawn and anal-
yzed. From the volume and composition changes
the volume of O2 uptake from or O2 output into the
pocket was calculated and plotted against the O»
tension in the pocket at that time. By interpola-
tion between 2 or more points one can determine
the O. tension in the pocket at which O; is neither
absorbed nor excreted and this is assumed to be
the tissue tension. CO: tension was measured
similarly. Tissue tension measurements thus ob-
tained were indistinguishable from steady state
pocket gas analyses. Furthermore, the similarity
of the pocket gas tension to expected venous blood
tensions would suggest that CO- and O tensions
of the pocket gas, the tissue and the venous blood
are practically equilibrated. (Supported by the
Air Materiel Command.)

625. Relative alterations of gastric iodide and
chloride clearances in dogs. L. VAN MipDLEs-
WORTH AND G. L. HowE.u.* Dept. of Physiology,
Univ. of Tennessee, Memphis.

The pylorus was cannulated and esophagus
ligated in Nembutal anesthetized dogs which

were fasted 6-18 hr. Twenty uc of carrier-free I)!

FEDERATION PROCEEDINGS

Volume 16

were injected intravenously, and 10-30 min. later
the stomach was washed repeatedly with water
until rinsings were free of mucus. At intervals of
15 min. gastric secretions were collected by wash-
ing the stomach 3-4 times, with about 300 cc water
per washing. Venous blood samples were obtained
at mid-point of each collection period. After 2
control collections animals were injected with
either histamine (0.1-0.14 mg/kg), NaSCN (80
mg/kg), or Nal (60 mg/kg). After 5-15 min., two
15-min. gastric collections were made and 2 blood
samples taken as before. Specimens were analyzed
for I'*! by gamma counting and for chloride by
HgNO; titration. In concentrations used, iodide
did not interfere with chloride determinations,
Control gastric clearances were: chloride 0.18
0.64 cc plasma/min. and iodide 2.6-10 cc/min. in
9 dogs. Following histamine, chloride clearance in-
creased an average of 144% but iodide increased
only 38%. NaSCN reduced chloride clearance to
57% of its previous value, while iodide clearance
was reduced to 11% of its control (iodide ap-
proached control chloride clearance). Nal de-
pressed gastric iodide clearance to 11% of controls
but chloride clearance was not significantly
changed. These data suggest at least 2 mechanisms
for gastric iodide secretion: a) a specific iodide
concentrating mechanism and b) a less specific
process, common to chloride and iodide. (Sup-
ported by grants from Atomic Energy Commis-
sion and Public Health Service.)

626. Failure of pregnane 3a,208diol and preg:
nane 36,208diol to inhibit estradiol-17
induced uterine growth and vaginal cornifi-
cation. JosepH T. VELARDO. Dept. of Anatomy,
Yale Univ. School of Medicine, New Haven, Conn.
Previously Hisaw, Velardo and Ziel (J. Clin.

Endocrinol. & Metab. 14(7): 763, 1954) reported

that pregnane 3a,208diol and pregnane 36, 208diol

were capable of inhibiting progesterone action in
decidual formation. This report is concerned with
the action of these 2 compounds on the uterine
growth promoting and vaginal cornifying activity

of 0.10 ug estradiol-178. Virgin, albino rats, 100

days of age, were bilaterally ovariectomized, and

1 wk. later 0.10 ug estradiol-178 was injected sub-

cutaneously daily for 3 days with each of the 2

pregnane compounds named above. All steroids

were injected at separate sites. Vaginal smears
were taken 24, 48 and 72 hr. after the initial in-
jections. Necropsies were performed 72 hr. after
the 1st administration of estrogen and the preg-
nane compound; the uteri were quickly removed
and weighed to the nearest 0.2 mg. Three daily
injections of 0.10 ug estradiol-17¢ effected uterine
weights of 227 mg (42.3 mg d. wt.). The simulta-
neous administration of 0.5-5.0 mg of pregnane
3a,208diol and 0.10 ug estradiol-178 produced

pee Sy a a oe

ga
ex]
lie

ime 1§

. later
water
vals of
wash-
- Water
tained
fter 2
| with
N (0
1., two
' blood
alyzed
ide by
iodide
ations,
» 0.18-
nin. in
nce in-
reased
ince to
arance
de ap-
al de-
ontrols
icantly
anisms
iodide
specific

(Sup-
mmis-

preg:
iol -178
ornifi-
vatomy,
, Conn.
. Clin.
ported
208diol
tion in
.d with
uterine
ctivity
ts, 100
1d, and
2d sub-
f the 2
teroids
smears
tial in-
. after
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e daily
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multa-
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oduced

March 1956

uteri that weighed 230 mg (42.0 mg d. wt.); 0.50-
5.0 mg of pregnane 38, 208diol likewise was without
effect in modifying the response of the uterus to
estradiol-178, the uteri weighing 234 mg (42.7 mg
d. wt.). These pregnane compounds did not alter
the vaginal cornifying action of estradiol; 95-100%
of the animals came into vaginal estrous 48 hr.
after the initial injection. When administered
alone, pregnane 3a, 208diol or pregnane 3, 208diol
did not modify the atrophic uterus or the vagina
of the ovariectomized rat. Data at hand suggest
the specificity of these pregnanes to restrict in
part progesterone whereas estradiol-178 activity
is not opposed.

627. Response of single medial geniculate
units to repetitive click stimuli. VERNON G.
VERNIER* AND RoBERT GALAMBOS. Walter Reed
Army Med. Cir., Washington, D.C.

The capacity of single medial geniculate units
to respond to repetitive click stimuli over a range
of frequencies was studied with KCl-filled micro-
pipettes oriented stereotaxically in unanesthe-
tized, paralyzed (Flaxedil) cats. Initially positive
spikes were recorded; the majority of units re-
sponded to clicks alone, fewer to noise alone and
still fewer to tones alone. Some responded to all
these stimuli but a moderate number of sponta-
neously active units were not aroused by any
sounds presented at moderate intensity levels.
Most click-sensitive units responded 70-100% of
the time when the clicks were presented at a rate
of 5/sec. at an intensity near the threshold for the
round window neural response. Above this rate of
presentation the percentage of clicks evoking
spikes declined smoothly, approaching zero some-
where above 20 clicks/sec. The click frequency at
which the unit responded 50% of the time varied
between 6 and at least 200/sec. in the 20 units
studied. These determinations seem to measure
some aspect of the excitability of the units exam-
ined. This aspect can be modified by certain drugs.
For example, morphine significantly increased the
click frequency at which response fell to 50% in
instances. It is possible that this technique might
be used for assaying drug effects upon the central
nervous system.

628. Some effects of protraction and frac-
tionation of dose of Co® gamma radiation
on 30-day mortality in the mouse. Howarp
H. VocEt, Jr., Joan W. CLark AND Donn L.
JorDAN (introduced by Austin M. Brugs).
Div. of Biological and Med. Research, Argonne
Natl. Lab., Lemont, Il.

When CF #1 female mice were exposed to Co
gamma-radiation (single, 90-min. whole-body
exposures), the 30-day lethal range was found to
lie between ca. 750 and 1100 r. These exposures

AMERICAN PHYSIOLOGICAL SOCIETY

193

were carried out in a gamma-neutron radiation
chamber, at dose rates between 8 and 12 r/min.
When the exposure time was increased from 1.5
to 24 hr., the 30-day Lbs of the mice increased
from 930 to 1325 r. In contrast, no such dose-rate
dependence was evident following similar expo-
sures of mice to fission neutrons. Mice were also
exposed (in the low-level Co® room at Argonne
National Laboratory) to a dose of 990 r, delivered
at the two dose rates of 11 r/min. and 3 r/min. A
significant difference in mortality was evident
following these intensities. From the data it is
postulated that approximately 3 of the ‘departure
from additivity’ previously reported for mice ex-
posed to mixtures of fission neutrons (3) and Co®
gamma rays ($) can probably be explained by the
lower dose rates of the gamma-radiation in these
mixture experiments. In order to compare effects
of dose protraction with those of fractionation,
experiments have been carried out as follows:
Paired doses of gamma rays, with intervals be-
tween irradiation of 24 hr., 48 hr. and 6 days will
be compared with single exposures in a study of
recoverable and irrecoverable components follow-
ing radiation. Similar fractionation exposures with
fission neutrons will be compared. The importance
of dose rate should be emphasized in the concept
of the relative biological effectiveness (RBE) of
two radiations. (Work performed under the aus-
pices of the U. 8. Atomic Energy Commission.)

629. Auditory cortical response to medial
geniculate stimulation. Curt von EvLER
(introduced by H. W. Macoun). Dept. of Anat-
omy, Univ. of California at Los Angeles, and VA
Hospital, Long Beach.

The auditory cortical response to single shock
stimulation of the medial geniculate body consists
of small initial deflections, a surface positive and
a subsequent surface negative wave. Both the
amplitude and the peak latency of the two latter
components are increased with 10/sec. stimulation
which may also introduce response variations.
Single shock responses recorded at successive
depths in the cortex show a diminution of ampli-
tude and an inversion of polarity of the main
response components, either at the turnover point
for click responses or at a more superficial level,
though never on passing through tiie surface
cortical layer. With repetitive stimulation, po-
tentiated responses may turn over in the same
manner, or may be obscrued by the recruitment of
components only recorded at the middle parts of
the cortex. The surface negative component of the
response to medial geniculate stimulation and a
surface negative response to direct cortical stimu-
lation close to the recording electrode interact with
initial occlusion, subsequent recovery and later
facilitation.

194

630. K-casein: micelle stabilizing protein in
milk. Peter H. von Hipret* anp Davin F.
Wauau. Dept. of Biology, Massachusetts Inst. of
Technology, Cambridge, Mass.

a- and #-caseins, which are defined essentially
on the basis of electrophoretic mobility and phos-
phorus content, have been obtained most fre-
quently from acid precipitated milk proteins. Re-
cently (J. Am. Chem. Soc. 77: 4311, 1955) we have
described a procedure for preparing a and £6 ¢a-
seins at constant pH (soluble casein) and charac-
teristic properties of a- and §-casein monomers.
If an attempt be made to reconstitute casein mi-
celles by adding calcium to an apporpriate mix-
ture of a and 6 caseins there results a granular or
flocculent precipitate of calcium caseinate, the
composition of which depends upon the tempera-
ture at which the calcium addition is carried out.
Characteristic micelles, as they occur in milk, do
not form. This is true both of soluble casein and
caseins obtained from acid precipitates. It was
found that during the process of fractionation of
a and £6 caseins (soluble casein) a component re-
sponsible for micelle stabilization had been re-
moved. This material which we designate k-casein
is a protein having an electrophoretic mobility
close to that of a casein; therefore it is expected
that its phosphorus content will be near that of
a casein. Its monomeric size is also close to that
already reported for a- and B-caseins; M ~ 13
to 25,000. It differs markedly from a and 6 casein
in a) its capacity to effect the stabilization of
casein micelles when present as the smallest frac-
tional component; b) the fact that polymerization
of k-casein alone is unaffected by temperature at
pH 7.0; c) the physical changes which take place
when k-casein is treated with rennin and d) its
capacity to effect the formation of a casein clot.

631. Pyrogen-induced fibrinolysis in man.
Kurt N. von KAvuLua AND JERRY WEIL (intro-
duce@ by JoserH Hotmss). Dept. of Medicine,
Univ. of Colorado School of Medicine, Denver.
Protein-free pyrogenic lipopolysaccharides pre-

pared by Westphal from S. abortus equ i(I) and E.

coli (II) were observed by Eichenberger to produce

fibrinolysis following injection in humans. In our

experiments, one injection of 0.2 ug I or 300 wg II

intravenously invariably induced strong fibrinoly-

sis in 21 humans. Smaller amounts produced vari-

able results. Fibrinolytic activity was detected 1

hr. after injection and lasted 3-6 hr., greatest ac-

tivity being 100-120 min. after injection and pre-
ceding the fever peak. Clot lysis at peak activity
required } hr. to several hours, and coagulograms
frequently revealed fibrinolysis beginning before
complete fibrin formation. The active fibrinolytic
specimens activated plasminogen, induced fibri-
nolysis in control plasma in dilutions of 1 part

FEDERATION PROCEEDINGS

Volume 1§

active to 19 parts control, and showéd increased
fibrinolytic action of their euglobulin fractions on
incubation. These findings indicated release of an
activator. The antiplasmin titer often became re.
duced, frequently after maximal fibrinolysis, and
the fibrinolytic activity of the urine showed major
fluctuations. After the disappearance of fibrinoly.
tie activity plasma samples often took longer to
lyse with urofibrinolysokinase, and euglobulin
fractions sometimes required more time for spon-
taneous fibrinolysis than before injection. Re.
duction of the febrile reaction with antipyretics
did not appear to diminish fibrinolytic activity,
Frozen plasma specimens lost their activity
slowly. Reinduction of fibrinolysis after 24 hr,
was possible; however, partial or complete re-
sistance usually developed after one injection. No
fibrinolysis could be induced by I and II in dogg,
rabits, or in vitro. (Aided by grants from the
American Heart Assn. and the Belle Bonfils
Bloodbank, Denver.)

632. Frontal corticofugal pathways for eye
movements. Irving H. Wacman, Howarp P,
KriEGER* AND Morris B. BENDER. Dept. of
Neurology, Mt. Sinai Hosp., New York City.
The frontal corticofugal pathways mediating

eye movements were studied in M. mulatta (encé-

phale isolé). The cortex and subcortex lying an-
terior to the motor strip were stimulated with bi-
polar electrodes oriented vertically using the
stereotaxic technique. The eye movement most
commonly elicited was conjugate, contralateral
deviation, usually horizontal, but occasionally
oblique. Ipsilateral movements were never ob-
tained. Eye centering and hystagmus occurred in-
frequently. All 3 movements were elicited from the
classic frontal eye field as well as from the adja-
cent posteromedial cortex. Stimulation of the
subcortex at various depths also yielded eye move-
ments. Analysis of these responses suggests that
the oculomotor pathways project from the cortex
posteromedially to the internal capsule. This is in
agreement with reported anatomic studies. Our
results disagree with those of Smith (In Precentral
Motor Cortex, edited by P. C. Bucy, p. 307, 1944)
and Crosby, Yoss and Henderson (J. Comp. Neu-
rol. 97: 357, 1952). Though differing in details
these authors suggest discrete cortical representa-
tion within the frontal area for each type of eye
movement. In contrast we obtained either hori-
zontal, upward or downward oblique deviation or
centering from the same point depending upon the
strength of stimulation. These discrepancies may
be related therefore to the differences in method,

e.g. anesthesia, parameters of stimulation. (Aided

in part by Public Health Service grant B-294

(C-2).)

ume 15

reased
Ons on
e of an
me re.
is, and
| Major
rinoly-
iger to
obulin
r spon-
n. Re-
yretics
tivity,
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ate re-
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ARD P,
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, 1944)
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Aided
B-294

March 1956

633. Compatative effects of intermittent com-
pression and of electric stimulation on de-
nervation atrophy. KHauit G. Waxim. Mayo
Clinic, Rochester, Minn.

Electric stimulation improves the work output
and endurance of denervated muscle and retards
the course of, but does not prevent, denervation
atrophy. This study compares the effects of elec-
tic stimulation and of intermittent compression
of denervated muscle to ascertain if the favorable
results of electric stimulation could be attributed
to intermittent muscular contraction and relaxa-
tion. Adult rats were divided into 4 groups.
Group 1 were controls. Groups 2, 3 and 4 underwent
excision of the sciatic and femoral nerves of one
extremity at the thigh. For 25-30 days, the de-
nervated legs in group 2 were stimulated elec-
trically (16 impulses/sec.) for 15 min. every half
hour throughout the 8-hr. day. Group 3 extremities
were stimulated similarly at 1 impulse/sec. In
group 4 the whole paralyzed limb was compressed
once a second by a cast containing a coil of thin
rubber tubing fitted over the extremity and con-
nected to a respirator that intermittently filled
the tubing with air at a pressure of 280 mm of
mercury. After this treatment, the tendo achillis
was separated from its insertion and connected
toa work-output machine to determine the initial
and total work output of the gastrocnemius-soleus-
plantaris muscles. The findings suggest that favor-
able effects of electric stimulation are not due to
intermittent contraction and relaxation of de-
nervated muscle. Intermittent mechanical com-
pression did not improve the work output and en-
durance of denervated muscles nor retard the
course of denervation atrophy.

634. Disturbances in neural metabolism re-
sulting from urinary products of phenyl-
ketonurics. Epwarp WALASZEK* AND L. G.
Asoop. Div. of Psychiatry, Univ. of Illinois
College of Medicine, Chicago.

Although many have speculated that the mental
retardation accompanying phenylketonuria may
be the result of toxic substances arising from an
inherent metabolic disorder, no experimental evi-
dence supporting such a hypothesis is available.
A crude ether extract of urine from phenylketo-
nurics was found to reversibly inhibit oxidative
phosphorylation (P/O) of rat brain mitochon-
dria in relatively small amounts. By means of
paper and ion exchange (Dowex 1) chromatog-
raphy, the ether extract was resolvable into at
least 3 components which were absent from nor-
mal urine. One compound was phenylpyruvic acid,
but the other 2 are as yet unidentified and may be
derivatives of indole. The R’s of the 3 compounds
with isoproponal: H2O:NH; (8:1:1) as the solvent
was no. 1—0.68 (phenylpyruvate), no. 2—0.78,

eae hae

AMERICAN PHYSIOLOGICAL SOCIETY

195

and no. 3—0.89. Phenylpyruvate at 10-* m pro-
duced about a 25% inhibition of P/O, while less
than 100 ug of no. 3 had a comparable inhibition;
no. 2 being somewhat intermediate in effect.
Known indole acetic acid at 10-4 m produced over
40% inhibition, while indole aldehyde was 5 times
as effective. A number of other indoles were tested
for activity and all were effective at 10-* m.
Evidence is available to suggest that such indole
derivatives can arise from a nonenzymatic oxida-
tion of urinary compounds such as dihydroxyphen-
ylalanine, e.g. the formation of 5-hydroxyindole.
(Supported by Office of Naval Research contract.)

635. Short P-R interval and fixed coupling in-
duced in dog by stimulation of sympathetic
nervous system. SHEPPARD M. WALKER. Univ.
of Louisville School of Medicine, Louisville, Ky.
Blood pressure and heart rate are markedly in-

creased in barbitalized dogs by injection of 0.06-

0.12 ce/kg of M/6 mixture of monobasic and di-

basic potassium phosphate (pH 7.6) into the

cisterna magna. The effects of strong stimulation
of the sympathetic nervous system, thus demon-
strated, were studied in vagotomized dogs. Such
stimulation induces short P-R intervals with pro-
longed QRS complexes showing fixed coupling with
the preceding beat. The R-wave of the prolonged
QRS complexes is slurred. The abnormal beat
usually alternates with a normal beat. The alterna-
tion may persist for 30 min. after an injection of
potassium phosphate. By peripheral stimulation
of the cut vagus nerve it is shown that the initial
segment of the prolonged QRS complex is not al-
tered when the preceding P-wave is abolished. The
usual alteration of the terminal segment of the
prolonged QRS complex by vagal stimulation is
regarded as evidence that this portion of the QRS
complex is due to a normally conducted impulse.

When the normally conducted impulse is blocked

by vagal stimulation, presumably, the ectopic

(initial) impulse invades the His bundle or one of

the bundle branches and thus completes the ex-

citation of both ventricles. Fixed coupling between
the preceding beat and the initial segment of the
prolonged QRS complex is a stable phenomenon,
not being disturbed during marked sinus arrhyth-
mia or during suppression of sinus rhythm by vagal

stimulation. (Supported by research grant H-

697(C5S) from the Natl. Heart Inst., Natl. Insts.

of Health, PHS.)

636. Relation between plasma volume and
vascular protein permeability. W. GorDoNn
WALKER AND Ricuarp §. Ross (introduced by
A. McGruee Harvey). Dept. of Medicine,
Johns Hopkins Univ. and Hosp., Baltimore, Md.
Disappearance curves of I'*!-labeled human

serum albumin and ['%-labeled human serum

196 FEDERATION PROCEEDINGS Volume i

gamma globulin were studied in 10 human subjects
during the 2-wk. period following intravenous in-
jection. The labeled materials were injected
simultaneously and the radioactivity of the
plasma samples resolved into its constituents on
the basis of the differing half-lives of the isotopes
used. Simultaneous disappearance curves for both
albumin and globulin have been obtained and
these curves analyzed to yield values for initial
protein distribution mass, plasma volume, total
exchangeable protein and ‘mean transcapillary
exchange rate.’ Thus, a comparison of simulta-
neous exchange rates and distribution volumes of
2 proteins of different size is possible in the same
subject. Simultaneous determinations of vascular
volume agree well (mean difference 8%). Albumin
and globulin exchange rates are linearly related
over the ranges observed. A comparison of the
vascular albumin mass with the extravacsular
albumin mass reveals a linear correlation (r =
+0.8), with the extravasuclar protein mass fall-
ing to zero when the vascular mass falls to 0.6
gm/kg. A similar relation exists between intra-
and extravascular globulin (r = +0.8). These find-
ings are consistent with a ‘barrier’ to protein
extravasation when the mass of vascular protein
is low. A possible explanation of the mechanism
involved is suggested by a comparison of the
plasma volumes with the ‘mean capillary exchange
rates,’ revealing that the rate constant decreases
as the plasma volume falls (r = +0.5, P < 0.02).
(Supported by Life Insurance Med. Research
Fund.)

637. Radioactive glucose and insulin hyper-
sensitivity in the hypophysectomized dog.
J.S. Wauu,* R. Steexe, R. C. pE Bopo ann N.
ALTSZULER.* Dept. of Pharmacology, New York
Univ. College of Medicine, New York City, and
Biology Dept., Brookhaven Natl. Lab., Upton,
Ae
In dogs in the postabsorptive state C' labeled

glucose administered intravenously by priming

dose followed by constant infusion maintained the
radioactivity of the plasma glucose at a constant
level. Glucose pool sizes and turnover rates were

calculated (STEELE et al. Federation Proc. 14: 286,

1955). While the C' glucose infusion continued,

glucagon-free insulin (0.025 u/kg., i.v.) was in-

jected. The variations of plasma glucose concen-
trations and radioactivity that followed insulin
permitted calculations of changes in glucose in-
flow from the liver into the rapidly equilibrating
part of the glucose pool and of glucose outflow
from this part of the pool. In both normal and
hypophysectomized dogs, although insulin caused
an initial decrease in inflow, the fall in plasma
glucose was due primarily to an increased out-
flow. The prompt restoration of the plasma glucose

level in the normal dog was caused by a suddenly
increased inflow. In the hypophysectomized dy
the increase in outflow was greater and the sudda
large increase in inflow did not occur, although
the insulin-induced hypoglycemia was _ greater
than in the normal dog. Maintenance of the hy.
pophysectomized dog on cortisone (0.8-1.5 mg/kg)
day) for several days resulted in an increase in its
glucose turnover rate to normal prior to insulin,
After insulin injection this animal exhibited
increase in outflow no greater than that observed
in the normal dog and a normal increase in its
glucose inflow in response to insulin-induced
hypoglycemia.

638. Galvanic skin reflex after acute spinal
transection in normal and decerebrate
eats. G. H. Wane (introduced by C. P. Riz.
TER). Labs. of Psychobiology and Otolaryngology,
Johns Hopkins Hosp., Baltimore, Md.

In a normal cat under anesthesia, stimulation
of a cutaneous nerve always evokes the galvanic
skin reflex from the foot-pads. It is abolished after
intercollicular decerebration. We have show
(Federation Proc. 14: 158, 1955) that its abolition
after decerebration is caused by the inhibitory
impulses from the bulbar ventromedial reticular
formation to spinal sudomotor neurons. In a nor-
mal cat, these bulbar inhibitory impulses are out-
balanced by the excitatory impulses from the
levels higher than the bulb. Severance of the
spinal cord that interrupts all its connection
with the brain removes the bulbar inhibitory im-
pulses in decerebrate cats, and excitatory and
inhibitory impulses, with the former dominating,
from all levels of the brain in normal cats. Conse-
quently spinal transection causes the reappearance
of the galvanic skin reflex in decerebrate cats and
a decrease in its intensity in normal cats. These
two types of results are obtained in the same cat.
Our results afford experimental evidence in sup-
port of Sherrington’s idea that spinal transection
produces its effects on reflexes through withdrawal
of the impulses from supraspinal levels of the
central nervous system.

639. Polarographic study of excreted com-
plexes of mercurials. I. M. WEINER AND
Orto H. Miuuer (introduced by O. W. Sar-
torius). Dept. .of Physiology, State Univ. of
New York, Upstate Med. Ctr., Syracuse.

In a previous report (J. Pharmacol. & Exper.
Therap. 113: 241, 1955) we have shown that dogs
excrete Salyrgan as a Scelyrgan-thiol complex.
In an extension of this study, we included another
mercurial, Neohydrin, and found that there are2
different thiols, thiols (I) and (II), respectively,
excreted in complexes with either mercurial. The
polarographic half-wave potential of the merecu-

Ma
rial

The
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March 1956

rial complex with thiol (I) is related to px in a
manner identical with that of mercurial-cysteine.
The half-wave potential-pH curve for the mer-
curial complex with thiol (II) differs in that it
lacks a characteristic inflection in the vicinity of
the pK of the amino group of cysteine. If cysteine
is a constituent of thiol (II) its amino group must
be tied up as it is in N-acetyl cysteine. After a
single intravenous dose of Salyrgan, thiol (I) is
the predominant thiol in the excreted complexes
for at least 40 min. but, 3 hr. after injection, thiol
(II) predominates. In similar experiments with
Neohydrin, thiol (I) predominates for the entire
3hr. In contrast, when either mercurial is infused
into the hepatic portal circulation, or when
Neohydrin is given orally, only thiol (II) is de-
tected in the excreted complex. Apparently thiol
(I) is derived from the kidney and thiol (II) from
the liver. Differences in tissue affinity for the
mercurials probably account for the time of ap-
pearance of thiol (II) after intravenous injection.
(Supported in part by a grant from the National
Insts. of Health, PHS.)

(40. Tissue oxygen consumptions of rats
adapted to cold. A. Kurt WEtss (introduced
by W. Guien Moss). Howard Hughes Med. Inst.
and Dept. of Physiology, Univ. of Miami School
of Medicine, Miami, Fla.

Cold exposure of a few days’ duration elevates
the resting metabolism of the intact rat. Various
tissues removed from such cold-adapted animals
have increased rates of oxygen consumption. The
extent of the elevation of the oxygen consumption
of these tissues varies with such factors as strain
of animal, age, sex, intensity and duration of cold
exposure. Generally. liver slices show the greatest
percentage increase; skeletal muscle comes next,
followed by cardiac muscle slices and diaphragm;
kidney slices, however, are apparently inconsistent
in their response. There are other tissues in the
cold-adapted rat whose oxygen consumptions are
not elevated in a statistically significant manner.
Brain cortex, lung, spleen, thymus and testis fall
into this latter category. The effect of cold
exposure on the oxygen consumption of tissues is
similar to the response to the administration of
Massive doses of thyroxine in thyroidectomized
tats which was demonstrated by Barker et al.
(Am. J. Physiol. 170: 81, 1952; Proc. Soc. Exper.
Biol. & Med. 83, 500, 1953). Therefore, the ob-
served effects of cold exposure on tissue metabo-
lism may have been partially induced by increased
thyroid activity. Additional factors have been sug-
gested which can probably modify the effects of
the thyroid gland on tissue metabolism; however,
these factors are apparently unable to elevate the
metabolism of those tissues which are refractory
to thyroid stimulation. (Supported in part by a

@venry

AMERICAN PHYSIOLOGICAL SOCIETY

197

research grant, RG-4150, from the National Insti-
tutes of Health.)

641. Separation of sphingolipides by adsorp-
tion chromatography. BENJAMIN WEISS
(introduced by Carney Lanpis). Depts. of
Biochemistry, New York State Psychiatric Inst.
and College of Physicians and Surgeons, Colum-
bia Univ., New York City.

Sphingolipides, isolated from monkey brain and
beef spinal cord according to the procedure of
Carter et al. (J. Biol. Chem. 169: 77, 1947), were
chromatographed on silicic acid columns and de-
veloped by gradient elution with chloroform and
methanol. The percentages of choline, galactose,
nitrogen, and phosphorus, and the choline:nitro-
gen, galactose: nitrogen, and phosphorus: nitrogen
molar ratios disclosed that glycosphingosides
formed bands I, II and III, and phosphosphingo-
sides, band V. Band IV, which consisted of 2 or 3
peaks, contained galactose and phosphorus and
gave positive Bial and ninhydrin reactions. On a
weight basis, bands I through V of monkey brain
sphingolipides comprised 4.4, 38.1, 24.5, 19.3 and
13.7%, respectively, of the total material recov-
ered. Overall recoveries averaged 94%. The upper
limit of resolution by the column was 4 mg of
sample per gram of packing. 1-C'4-Octanoic acid,
chromatographed with unlabeled sphingolipides,
emerged with band I. When labeled sphingolipide
preparations, obtained from monkey brains per-
fused with 1-C14-acetate or 1-C14-octanoate, were
dialyzed and then chromatographed, the C™
activity resided exclusively in band IV. (Sup-
ported by research grant B-344 from the Natl.
Inst. of Neurological Diseases and Blindness,
Natl. Insts. of Health, PHS.)

642. Effect of age, gonadectomy and gonado-
trophins on blood pressure of chicks. H. 8.
Weiss,* R. K. Rincer* anp P. D. Strurxie.
Rutgers Univ., New Brunswick, N.J.

The systolic pressure of adult male chickens and
capons averages 20% higher than that of the fe-
males and can be depressed to near female level by
estrogen suppression of pituitary gonadotrophins
(STURKIE AND RincEerR. Am. J. Physiol. 180: 53,
1955). Since the development of the sex difference
in pressure had not been fully explored in imma-
ture birds, consecutive monthly measurements
were undertaken on intact and gonadectomized
males and females from 3 wk. to 10 mo. of age.
Birds from each group were also treated with gona-
dotrophin (PMS) between the 7th and 9th wk.
Under 8 wk. of age there were no pressure dif-
ferences between the sexes. Starting between 8
and 12 wk., male systolic pressure rose rapidly
and within 4-5 wk. attained its characteristic
adult level. Female pressure remained relatively

198

stable, apart from seasonal fluctuations. Hemo-
dynamic changes in the male over a 4-wk. interval
during which the major sex difference in pressure
was developing were: systolic, +21 mm; diastolic,
+7 mm; pulse pressure, +14 mm; heart rate,
—35/min. Poulard and capon pressures paralleled
the rise of those of the males, but were delayed 4
wk. Thus the pressure of the chick climbs to the
adult male level unless modified by ovarian ac-
tivity. Lack of testicular activity apparently de-
lays the initiation of the rise. No pressure change
followed PMS administration, despite marked
reproductive stimulation, indicating that neither
gonadotrophin nor sexual development is solely
responsible for the rise in pressure.

643. Effects of chronic hyperventilation upon
hypocapnic tolerance. J.G. WELLS, B. BALKE
AND J. P. Exuis, JR. (introduced by Hans G.
CLAMANN). Dept. of Physiology-Biophysics,
USAF School of Aviation Medicine, Randolph
Air Force Base, Texas.

There are numerous reports that suggest that
there are various tolerances to hypocapnia pro-
duced by hyperventilation. In this study 6 normal
males received 5 exposures to a standardized
laboratory hyperventilation test. Hypocapnia
was introduced by means of a positive pressure
respirator. Following the control experiment,
tests were administered after hyperventilation
training; a 2}-wk. period of daily exposures to
overventilation, an 8-wk. physical training pro-
gram during which time the subjects were not
mechanically hyperventilated, a 6-wk. residence
at 14,160 ft. and following return to ground level.
The control test was terminated after 30 min. as
the subjects developed severe symptoms of hypo-
capnia. The hyperventilation training resulted
in an adaptation which permitted extension of
testing time to 60 min. with an elimination of
symptoms. Initially psychomotor performance, as
determined on a SAM multiple dimensional
pursuitmeter, was reduced to 49%; however, in
subsequent tests efficiency was above 70%. Res-
piratory data, ventilation (1/min.) and alveolar
Pco, (mm Hg), presented a similar pattern in that
following the period of daily exposures to hyper-
ventilation the subjects tolerated an increased
ventilation and a decreased Paco, without de-
veloping hypocapnic symptoms. Terminal values
in the control test were ventilation 39.0 and
Paco, 16.0. In the tests that followed ventilation
was approximately 50 witha Paco, of 12.5 with the
exception of the test at altitude which resulted in
79% efficiency, 53.6 l/min. and 9.3 mm Hg for
psychomotor performance, ventilation and al-
veolar Pco., respectively.

644. Effect of embolization via coronary ar-
terial catheterization on coronary blood

FEDERATION PROCEEDINGS

Volume 1§

flow and coronary sinus blood oxygen

saturation. J. W. West, T. Kosayasn,

L. ScHLesincER, F. 8. ANDERSON AND S. M,

BaNNETT (introduced by C. F. Scumipy),

Dept. of Pharmacology, Univ. of Pennsylvania,

School of Medicine, Philadelphia.

The effect of coronary embolization was studied
in intact dogs anesthetized with sodium pento-
barbital. Under fluoroscopic guidance, a metal
cannula was placed in the ascending aorta near the
coronary ostia or specially designed catheters were
introduced into the left circumflex and left an-
terior descending coronary arteries via the carotid
artery. Similarly a modified Morawitz cannula was
inserted into the coronary sinus via the right
jugular vein. Coronary blood flow and the 0,
content and capacity of coronary sinus and ar-
terial blood samples were determined. Prior to
embolization, direct catheter injections of acetyl-
choline and veratridine were utilized in verifica-
tion of the positions of the catheters. Positions of
all catheters and cannulae were confirmed by
autopsy after each experiment. Arterial injections
of lycopodium spore suspension, of 0.1 cc of 5%,
in the ascending aorta, or 0.08 cc-0.13 ce of 1%,
into the left circumflex or anterior descending
coronary artery, caused an immediate decrease of
approximately 30% in coronary flow with a fall in
coronary venous oxygen saturation of the sinus
blood lasting approximately one minute, followed
by an increase of approximately 65% in coronary
flow with a rise in coronary venous oxygen satura-
tion, occurring within 5 min. and lasting for ap-
proximately 5 min., provided blood pressure was
not appreciably decreased. The initial decrease of
flow and saturation was expected from the ob-
struction of the coronary bed by the emboli; the
subsequent increase in coronary blood flow was
totally unexpected. Cutting all extrinsic nerves
of the heart did not eliminate the response.

645. Effect of exercise on splanchnic blood
flow and splanchnic blood volume in man.
Henry O. WHEELER,* OWEN L. WapeE,* Burton
ComBEs,* ALFRED W. CuiLps,* ANDRE CouR-
NAND AND STANLEY E. Bran ey. Dept. of Medi-
cine, Columbia Univ., New York City.

The effect of exercise (alternate leg raising) on
splanchnic blood flow (EHBF-Bromsulfalein
method), splanchnic blood volume (SBV-regional
dilution of I'*! labeled human serum albumin),
total blood volume (I'*! albumin), and total and
splanchnic oxygen consumption was studied in
five convalescent male patients. EHBF fell in all
subjects during exercise by 240-460 ml/min. and
returned during recovery toward control levels in
all but one. Resting SBV averaged 1160 ml (17-
25% of total blood volume) and decreased during
exercise in all subjects by 285-700 ml (mean 400

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March 1956

ml) so that 6nly 10-15% of total blood volume re-
mained in the splanchnic bed. During recovery
SBV increased in all subjects but not to the con-
trol levels in three. During exercise total oxygen
consumption increased. Splanchnic oxygen con-
sumption fell slightly in all subjects but rose above
control levels during recovery. Since blood pres-
sure tends, if anything, to increase during exer-
cise the reduction in EHBF indicates splanchnic
vasoconstriction. This response serves to supple-
ment blood flow to active muscle by diverting
blood from the splanchnic bed. The decrease in
SBV represents a significant ‘autotransfusion’ of
blood into the general circulation. As a result,
venous return may be augmented early in exer-
cise, thereby facilitating a more prompt increase
in cardiac output.

646. Some biochemical effects of dl-methio-
nine, including depressed renal transport
of paraaminohippurate (PAH). ABRAHAM
G. Wu1TE. Dept. of Medicine, The Mount Sinai
Hosp., New York City.

Studies were performed on the effects of dl-
methionine upon the: a) acetylation of PAH by
pigeon-liver mince, b) oxygen consumption of
ethanol-respiring yeast, c) uptake and acetylation
of PAH by rat renal slices, and d) stimulatory ef-
fect of acetate on the uptake and acetylation of
PAH by rat renal slices. dl-Methionine (8mm/1.)
depresses the acetylation of PAH by pigeon-liver
mince and by rat renal slices to 52.2% and 43.1%
of their control values, respectively. The stimu-
latory effect of 0.01 m acetate on the acetylation
of PAH by rat renal slices is inhibited completely
by dl-methionine (16 m/l). d-Methionine (8
m/l.) inhibits the acetylation of PAH by pigeon-
liver mince, while /-methionine (8 mm/I.) is com-
pletely inactive in this respect. The oxygen con-
sumption of ethanol-respiring yeast is reduced to
62.9% of the control value by dl-methionine (8
ma/1.). dl-Methionine (8 mm/1.) depresses the up-
take of PAH by rat renal slices to 27.1% of the
control value; the J-isomer has no depressant ef-
fect. dl-Homocysteine and dl-cysteine (16 mm/1.)
each depress the acetylation and uptake of PAH
by rat renal slices, while dl-valine and dl-alanine
have no depressant effect. dl-Methionine inhibits
the stimulatory effect of acetate on the renal trans-
port of PAH. Possible mechanisms underlying
action of dl-methionine (disruption of citric acid
cycle and depression of Coenzyme A activity) are
discussed. Data on the transport and acetylation
of PAH by guinea pig slices indicate that the
transport of PAH by such slices is two-directional.
(Aided by PHS grant H-1245.)

47. Possible subcortical mechanisms requi-
site to forced circus movements. RICHARD

AMERICAN PHYSIOLOGICAL SOCIETY

199

P. Wuite* anp Harop E. Himwicn. T'hudichum
Psychiatric Research Lab., Galesburg State Re-
search Hosp., Galesburg, Ill.

This paper describes results obtained from elec-
trophysiologic, extirpational and biochemical
studies on the phenomenon of forced circling. Pre-
vious workers have shown that the injection of
diisopropylfluorophosphate (DFP) into the right
common carotid artery of the rabbit may induce
either counter-clockwise or clockwise forced cir-
cus movements. Decortication prior to the injec-
tion of DFP did not prevent these two basic forced
circus movements, a finding which indicates the
possible importance of subcortical centers to this
phenomenon. Electrostimulation with bipolar
electrodes (0.8-1.2 v., 5.0 msec. duration) im-
planted within the right mesodiencephalic region
produced clockwise circus movements (tegmental
response) in unanesthetized rabbits. Conversely,
bipolar stimulation of the right caudate nucleus
(1.0-1.5 v., 5.0 msec. duration) elicited counter-
clockwise circling which could not be duplicated
by stimulation of adjacent white matter or the
cerebral cortex. Moreover, the injection of DFP
(0.06-0.16 mg) directly into these same structures
by means of a micrometer syringe produced similar
results. Cholinesterase activity determinations
reveal that DFP so administered is confined prin-
cipally to the site of injection. Therefore, it seems
reasonable to postulate that the 2 diametrically
opposite effects often seen with unilateral arterial
injection of DFP may be due to preferential stimu-
lation of either basal ganglion or mesodiencephalic
structures. Thus, the subsequent ablation of the
right caudate nucleus of an animal circling count-
er-clockwise as a result of an arterial injection of
DFP usually stops entirely this circling. Similarly,
destruction of the right rostral tegmentum will
stop clockwise circling.

648. Monomyofibroscopy: effect of disodium
adenosine triphosphate and sodium uridine
triphosphate on the shortening of muscle
protoplasm. Fioyp J. Wiercinski. Dept. of
Physiology, Hahnemann Med. College, Phila-
delphia, Pa.

Monomyofibroscopy I define as the study of one
or more isolated muscle fibers obtained from a
fresh muscle immersed in an isotonic solution and
observed under the microscope. Various methods
of experimentation can be applied and an adequate
control is essential. The method used in the present
study is the microinjection of solutions into muscle
fibers obtained from the adductor magnus muscle
of the frog immersed in a 0.12 mM NaCl solution at a
pH of 6.0. Solutions were prepared from a fully
crystalline form of disodium adenosine triphos-
phate, (ATP), monosodium uridine triphosphate
(UTP), and a combination of the two salts. Fresh

200

fibers were injected until a trace of the solution
could be seen along the entire length of a piece of
fiber. The effect on the shortening of the fiber was
observed. The results are stated as an average per-
centage of shortening. Care was taken to inject
the fibers with uniform pressure, since variations
in technique can influence the results. Quartz
redistilled water at pH 6.0 gave a result of 12%.
A 0.12 m NaCl solution isotonic with muscle in
most cases caused very little shortening of 8%.
When the fiber is injected with isotonic solution
at a high pressure the shortening may be as much
as 19%. A 0.005 m ATP solution adjusted to 0.1
mM NaCl and pu 6.1 gave a result of 12%. A 0.005 m
UTP solution adjusted to 0.1 m NaCl and pu 6.1
gave a result of 10%. A combination of 0.005 m
ATP and UTP solution adjusted to 0.15 m NaCl
and pH 6.5 gave a result of 15%. These data sug-
gest that the microinjection of a solution prepared
with crystalline disodium adenosine triphosphate
or sodium uridine triphosphate in small concen-
trations under the conditions of the experiment
produced little or no effect on the shortening reac-
tion of muscle protoplasm.

649. The piscine heart. CHARLES G. WILBER.
Chemical Corps Med. Labs., Army Chemical Ctr.,
Md.

Two species were studied, Fundulus heteroclitus
the killifish and Opsanus tau the toadfish. Heart
rates were recorded electrocardiographically. Dar-
stine, an atropine-like drug, was administered in-
traperitoneally in various doses to each species.
A dose of 20 ug in the killifish elicited no observa-
ble response, 100 ug resulted in darkening of the
fish within 5 min. Doses greater than $ mg were
lethal. The heart of Fundulus was markedly accel-
erated by darstine. For example, one specimen
had a control rate (20°C) of 70 beats/min.; 30 sec.
after giving $ mg of darstine the rate jumped to
140; after 45 sec., the rate was 170 and remained
at that level for 5 min. In the toadfish no color
changes were observed after darstine even after
doses of 20 mg. The control heart rate (20°C) in a
typical specimen was 20-30 beats/min. It was un-
changed by the highest dose of darstine used.
Atropine sulfate, 20 mg/fish, also failed to modify
the heart rate of the toadfish, although in Fundu-
lus atropine accelerated the heart as did the dar-
stine. Decamethonium given intraperitoneally in
doses as high as 500 mg had no measurable effect
on the toadfish heart. If 1 mg of decamethonium
was injected into the killifish, a decreased heart
rate resulted. In a typical specimen, the control
rate was 150; 1 min. after the drug was given it had
dropped to 95; 8 min. after treatment it fell to 82
where it remained for an hour. It is apparent that
Fundulus has a heart which responds readily to
drugs; the toadfish heart is quite resistant to drug

FEDERATION PROCEEDINGS

Volume ij

action. The reason for these differences is obscure
at the moment but may be related to difference
in vagal influence in the 2 species.

650. Chemical analyses of post-mortem tis.
sues as aid in determining physiological
status of flying personnel prior to aircraft
accidents. S. S. Witks, DonaLp D. Van Fos.
SAN AND Rosert T. Cuark, JR. (introduced by
E. A. Buarr). Dept. of Physiology-Biophysics,
USAF School of Aviation Medicine, Randolph
Air Force Base, Texas.

A new technique has been developed which pro.
vides a means for determining the CO saturation
level of blood by analyzing tissues for CO content
and correlating with the blood CO level. A series
of 280 rats and 10 dogs were used in establishing
the relationship between CO blood level and C0
content of tissues. CO determinations were made
on various rat tissue when the animals were
treated as follows: 1) breathing different CO con-
centrations and killed by decapitation; 2) normal
animals killed (decapitation, trauma, drowning)
and subsequently burned; 3) animals killed in
burning gasoline and jet fuel; 4) animals exposed
to CO and subsequently burned to death, and 5)
post-mortem tissue (control and CO-tissue) ex-
posed to natural environment for periods of 8-66
hr. The investigative data show that the blood CO
levels can be determined by tissue analysis. At a
50% blood COHb level the tissue CO values in
cc/100 gm were: muscle, 0.075; liver, 0.245; grain,
0.0857; kidney, 0.250; lung, 1.190; heart, 0.572. In
the analysis of blood of 130 rats for CO the values
obtained by the homogenizer and the Van Slyke
methods agreed within 0.3 vol. %.

651. Renal excretion in a fresh-water turtle.
James K. WixutaMs (introduced by Louis A.
Toru). Dept. of Biochemistry, Louisiana State
Univ. School of Medicine, New Orleans.

The urine composition of the turtle Pseudemys
scripta elegans was investigated. Under varying
conditions of hydration the turtle produced urine
whose concentration varied from a low of 5 mOs/L
to a high which was approximately equal to the
osmotic pressure of the plasma. The average urine
composition of the normal fasting turtles imme-
diately after removal from a tank of water was a8
follows: NH;—8 ME/I., CO.—12 ME/I., Cl-3
ME/1., Na—3 ME/l., K—6 ME/I. and Urea-82
mM/l. The median pH was 6.8, and the average
osmotic pressure was 92 mOs/]. Dehydration de-
creased and hydration increased the osmolar
clearance without affecting the ratio of the urinary
components. The injection of CaCl. produced an
acidosis which increased the loss of Cl without sig-
nificantly affecting urinary NH;. NaHCO; caused
a decrease in urinary NH; and an increase in Na

eerrr

olume 16

obscure
Terences

em tis.
rlogical
aircraft
AN Fos.
uced by
physics,
randolph

ich pro-
buration
content
A series
blishing
and C0
re made
Is were
SO con-
normal
owning)
illed in
exposed
_ and 5)
ue) ex-
of 8-66
ood CO
is. Ata
ues in
; grain,
572. In
: values
1 Slyke

turtle.
yuis A,
a State

udemys
rarying
d urine
mOs/L
to the
e urine
imme-
was a8

cl-3
Jrea-52
verage
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inary
ced an
ut sig-
caused
in Na

March 1956

and CO2. The injection of the carbonic anhydrase
inhibitor, 6063, resulted in the excretion of an
alkaline urine which contained K and CO:z in high
concentration. The rise in K produced a fall in
NH;. It is concluded that the turtle resembles
man in its reaction to 6063, and that it resembles
the alligator in its failure to increase NH; excre-
tion in acidosis (Proc. Soc. Exper. Biol. & Med.
88: 682, 1955).

652. Oxygen uptake of adrenal slices from
testectomized rats of different ages. MARTIN
W. WriuiamMs* anp C.uirrorp A. ANGERER.
Dept. of Physiology and Biophysics, Univ. of Ver-
mont, Burlington, and Dept. of Physiology, Ohio
State Univ., Columbus.

The oxygen uptake of adrenal slices from 90-
and 180-day white rats was studied by the Fenn
microvolumetric technique. Testectomy was per-
formed at 20-24 days of age. Eighty-six testec-
tomized and 98 control rats were used. Aliquot
samples of pooled adrenal slices were studied in
phosphate buffered (px 7.4) Krebs solution. After
the Ist hour, a given substrate (citrate, glucose,
lactate, pyruvate and succinate) into the tissue-
Krebs solution to give a final arbitrary concentra-
tion of 200 mg %. Comparisons of adrenal mean
Qo, values for 90-day testectomized and control
animals were not significant (Student’s method)
for citrate and glucose, but were significant for
Krebs —22.5% (P < .01), succinate +20% (P <
05), lactate —50 (P < .01) and pyruvate —35.8%
(P < .01). Significant differences in Qo, values
were also noted between the 180-day testectomized
animals and their controls: Krebs —18.2% (P <
05), citrate — 36.1% (P < .05), and succinate
-22.5% (P < .01). Cross comparisons of Qo,
values for 90- and 180-day old control groups
showed significant ageing effects: Krebs —35.6%
(P < .01) and succinate +32.1% (P < .01). No
significant difference was noted for citrate. Simi-
lar comparisons from the same age groups, but
testectomized, indicate the following aging effects:
Krebs—32.4% (P < .01), citrate —51.4% (P < .01)
and succinate —14.7% (P < .02).

653. Inhibition by vitamin By of cortisone-
induced weight loss in mice. W. LANE WIL-
uams. Dept. of Anatomy, Univ. of Minnesota,
Minneapolis.

Cortisone (2.5 mg daily, subcutaneously, for 7
days) was administered to 100 young adult mice.
Water and the standard ration (Fox Chow) were
available to these mice at all times. Of these mice,
50 also received daily subcutaneous injection of
lug of vitamin By for 7 days. Weight loss was
11.8% in the cortisone group, and 5.3% in the cor-
tisone plus Bye group. Cortisone (1.25 mg daily for
3 days) was administered similarly to 100 fasting

AMERICAN PHYSIOLOGICAL SOCIETY

201

mice. Fifty of these mice received concurrent in-
jections of 2 ug of vitamin By. Within 3 days those
injected with cortisone (alone) lost 28% of body
weight. The cortisone plus Biz group’s weight loss
was 24% (1% less than that in 50 control-starved
mice not injected with cortisone). Fifty starved
control mice injected as above with vitamin Bi
(but not with cortisone) showed a weight loss of

20%.

654. Effect of blood pH and bicarbonate upon
renal reabsorption of calcium. BILLY JAMES
WiLLIAMsON* AND SmitTH FreEMAN. Dept. of
Biochemistry, Northwestern Univ. Med. School,
Chicago, Ill.

The effect of acute disturbances in acid-base
balance upon renal mechanisms of calcium excre-
tion was studied in adult female dogs. Creatinine
was used to measure GFR. Filtration, reabsorption
and excretion of the following ions were measured:
calcium, potassium, sodium, phosphate, bicar-
bonate and citrate. Filterable calcium was deter-
mined at controlled pH by means of a parlodion
membrane and CO:2-O2 gas mixtures under pres-
sure. Blood and urine pH and total CO: values
were obtained with each set of determinations
and the H:CO; and HCO;- calculated. In each
experiment, after normal values were established,
observations were made on one or more of the fol-
lowing conditions: respiratory acidosis, metabolic
acidosis, metabolic alkalosis and compensated
metabolic alkalosis. Calcium excretion was in-
creased in all conditions, though most markedly
in metabolic alkalosis and its compensated state.
The plasma concentration of total calcium was
decreased in metabolic alkalosis and its compen-
sated state; the filterable fraction, however, re-
mained at a normal level except in metabolic
acidosis where it was increased. The GFR was
normal in alkalosis, but increased in the other
states. Reabsorption of calcium/100 ce GFR was
inversely proportional to blood px and/or plasma
bicarbonate level; it was inversely proportional
also to sodium excretion/minute. Calcium loading
experiments were performed in controls and in
metabolic alkalosis. No evidence of a Tm or of an
active tubular secretion for calcium was demon-
strated. Citrate excretion was directly propor-
tional to the urine pH. (Supported by a research
grant, No. A-666, from the PHS.)

655. Effect of intra-arterial injections of epi-
nephrine on spinal flexor and extensor re-
flexes. Victor J. Witson (introduced by D.
McK. Riocu). Walter Reed Army Med. Cir.,
Washington, D. C.

The effect of synthetic pure J-epinephrine on
spinal reflexes was studied in decerebrate, acute
spinal and chronic spinal cats. Intra-arterial in-

202 FEDERATION
jection was into the abdominal aorta by way of the
left renal artery. Injection of 5-40 ug/kg increased
monosynaptic extensor reflexes evoked by single
shocks in acute and chronic spinal preparations.
Sustained extensor reflexes were increased in
chronic spinal animals, but modified with great
variability in decerebrate preparations. The in-
crease produced by epinephrine in extensor re-
flexes lasted several minutes, with a peak 4-1 min.
after start of injection. Low doses evoked only
increases in extensor activity. Higher doses often
caused more complicated effects such as a brief
early depression, a period of enhancement, and a
late, sometimes irreversible knockdown. The
latter seems due to excessive amounts of the drug.
Monosynaptic and polysynaptic flexor reflexes
were diversely affected by epinephrine. No evi-
dence for a consistent reciprocal action on exten-
sor and flexor reflexes (as described in SkKoGLUND,
Cold Spring Harbor Symp. 17: 233, 1952) was ob-
tained. Changes in the size of polysynaptic re-
flexes were accompanied by latency changes, sug-
gesting a possible drug action on interneurons.
The action of epinephrine on spinal reflexes was
not correlated with appreciable changes in sensory
input to the cord and was independent of systemic
blood pressure changes. The action of the drug on
the central nervous system seems to be a fairly
general one, involving modification of some factor
determining cellular excitability.

656. Tremor in African green monkeys. W. F.
WINDLE, J. CAMMERMEYER,* E. R. Frerinea,*
JANE JORALEMON,* J. O. SMartT* anp M. P.
McQu1LuEN.* Lab. of Neuroanatomical Sciences,
Nail. Insts. of Health, Bethesda, Md.

Tremors associated with other phenomena were
induced by harmine, harmaline, A-9138 (Abbott
Labs.), chlorpromazine and reserpine. Harmine,
10 mg/kg subcutaneously, within 25 min. produced
appeargnce of apprehension, hyperirritability,
hyperactivity and coarse tremor; at 15 mg/kg
these phenomena appeared in 15 min. and were
followed immediately by convulsions. Harmaline,
15 mg/kg intraperitoneally, within 13 min. induced
hyperirritability, impaired balance—locomotion
on wide base— and coarse tremor which ceased
during voluntary movements. A-9138, 5-15 mg/kg
subcutaneously, produced intense visceral crises
(diarrhea, urination, cutaneous secretion, lacri-
mation, copious salivation) in 30-60 min.; the ani-
mals appeared apprehensive and displayed, alter-
nately, phases of hyperactivity and lethargy.
Coarse tremor during voluntary effort appeared
in 2-3 hr. Banthine and atropine only temporarily
controlled visceral effects. Symptoms at all doses
persisted 20 hr. or more. Monkeys receiving 10-15
mg/kg showed hypalgesia, esotropia, hypokinesia,
lethargy and extreme prostration on the second

PROCEEDINGS Volume ij
day, one dying in cardiac arrest. Chlorpromazine,
20 mg/kg intramuscularly, induced hypokinegig
and somnolence in 15-30 min.; fine tremor ap-
peared in 40 min., followed 1 hr. (during recovery
from lethargy) by coarse resting tremor. Reger.
pine, 2.5 mg/kg orally or intravenously, led
to akinesia, rigidity of ‘cogwheel’ type, coarge
tremor at rest, sialorrhea, changes in mood and
expression. Symptoms appeared in 30 min., maxi-
mum effects in 2-4 hr. and recovery in 24-48 hr,
Chronic daily reserpine, 0.2-0.4 mg/kg subeu-
taneously, induced hypokinesia, slight rigidity
and marked coarse tremors without the vegetative
phenomena. Effects were reversible.

657. Enhanced carbohydrate oxidation in ad.
renalectomized rats. W. W. WINTERNITz (in-
troduced by D. I. Hitcucock). Dept. of Physi-
ology, Yale Med. School, New Haven, Conn.
Previous studies have demonstrated a deficit of

carbohydrate in adrenalectomized rats following

epinephrine (Proc. Soc. Exper. Biol. & Med. 81:

683, 1952). Further experiments were undertaken

to determine the fate of this carbohydrate. Re-

spiratory quotients were determined of fasted
adrenalectomized and control rats at intervals
following subcutaneous epinephrine administra-
tion. The normal rats showed a small increase in
carbohydrate oxidation as measured by the respir-
atory quotients. In contrast, the adrenalectomized
animals showed a high peak of carbohydrate oxida-
tion in the 2nd and 8rd hours after epinephrine.

Calculations indicate that 80-90 mg carbohydrate/

100 gm rat are oxidized by the adrenalectomized

rat in excess of that oxidized by the normal during

the 3-4 hr. following epinephrine administration.

This accounts for a large part of the above-men-

tioned carbohydrate deficit in adrenalectomized

rats. It suggests that the adrenal cortical hor-
mones are concerned with the inhibition of carbo-
hydrate oxidation in the liver, since Wick and

Drury found no difference in oxidation of carbo-

hydrate by adrenalectomized eviscerated animals

as compared to normals.

658. Skin temperature changes during pro-
duction of experimental frostbite. ROBERT
A. Woxsacu,* JosepH R. Buarr, Tuomas B.
Roos* anp Wiiu1aAmM F. Srravuss.* Dept. of
Physiology, Harvard Medical School, Boston,
Mass.

Cold exposure produces alternate vasoconstric-
tion and vasodilatation in man (Lewis, 1929).
Skin temperature changes indicate similar phe-
nomena in the rabbit. Unanesthetized rabbits, im-
mobilized on a canvas hammock with feet depend-
ent, were exposed at —15° to —25°C. Rectal tem-
perature fell slightly but was maintained above
37.5°C with electric heating pads. One or both

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March 1956

hind feet were depilated and uninsulated. After
1-5 hr. exposure freezing usually occurred, and
fourth degree frostbite developed subsequently.
Skin temperatures were recorded from the four
distal phalageal toe pads (Toe T) and dorsal to
the metatarsal heads (Met T). At the onset of cold
exposure Met T fell rapidly from 30° to 35°C. (5-
10° above room temperature) toward 0°C. From
temperatures between —2.5°C and +3.5°C, Met T
frequently rose more than 3°C. Such temperature
increments were typically more frequent, of
greater amplitude and of shorter duration during
-25°C exposure than during —20°C exposure. Toe
T changes were more marked than Met T changes.
Supercooling, frequently observed in the toes, was
less common in the metatarsals. When vasodilata-
tion ceased, Met T fell to approximately —1°C,
then gradually to —2°C, indicating onset of freez-
ing, and finally more rapidly toward ambient
temperature. After cold exposure, thawing in room
air was usually complete within 45 min. Skin tem-
peratures remained below room temperature up to
100 min. after thawing, indicating persistent vaso-
spasm. Thawing by 10 min. immersion in 42°C
water did not markedly lessen the duration of
vasospasm. (Supported by Contract DA49-007-
MD-342 with the Office of the Surgeon General,
Department of the Army.)

659. Effects of controlled exercise on experi-
mental atherosclerosis in androgen-treated
chicks. Harry Y. C. Wonea, Rosert L. Sim-
MONS AND Epwarp W. HawrTuorne (intro-
duced by JoserH L. Jounson). Dept. of Physi-
ology, Howard Univ. College of Medicine, Wash-
ington, D.C.

Previous observations in our laboratory (Am.
J. Physiol. 178: 269, 1954) indicate that exercise
depresses the therapeutic effects of androgen
(comb growth) in capons. The present investiga-
tion was undertaken to study the effect of testos-
terone propionate and exercise in cholesterol-fed
cockerels. Four groups of cockerels were studied:
1) controls, those receiving no therapy, cholesterol
or exercise; 2) no exercise but cholesterol-fed; 3)
treated with 2.5 mg of testosterone propionate
daily plus cholesterol but not exercised; 4) simi-
larly treated as group 3 with the exception that
these birds were exercised twice daily for 8 wk.
The ‘atherogenic’ diet consisted of 2% cholesterol
and 5% cottonseed oil with mash. After 8 wk. on
on this regimen, results obtained were as follows:
1) the incidence of abdominal atherogenesis was
none in group 1, slightly higher in group 4, and
highest in groups 2 and 4; 2) chicks treated with
2.5 mg testosterone propionate daily on a choles-
terol diet, whether exercised or not, had a signifi-
cantly lower blood cholesterol level than birds on
cholesterol alone in comparison to the controls.

AMERICAN PHYSIOLOGICAL SOCIETY

203

Our data indicate that exercise does not reduce the
blood cholesterol level of androgen-treated birds
on an ‘atherogenic’ diet. However, it seems that
exercise may have a marked effect on the informa-
tion of atheromatous plaques in the abdominal
aorta of young chicks.

660. Effects of absorbency of clothing mate-
rials. ALAN H. Woopcock anp RicHarp L,
Pratr.* Quartermaster Research and Develop.
Ctr., Natick, Mass.

Textiles of similar structure made of wool and
nylon were conditioned overnight in atmospheres
of both high and low humidities. When placed on a
copper plate whose temperature was maintained
at a constant level, the heat supply was measured
until equilibrium was established. After condi-
tioning at high humidities, the woolen textile
showed a markedly higher heat loss than the nylon
for about 24 hr. At low humidities the wool con-
tinued to show a higher heat loss than nylon but
the effect was less marked and persisted for a
shorter time. There was some effect of the humid-
ity change on the nylon which absorbs up to 5%
moisture, but it was negligible compared to the
wool. Differences in heat loss are due to the heat
of absorption of water which can be considered as
adding to the heat capacity of the clothing. Ab-
sorption and desorption are transient effects which
occur when conditions are changed. If conditions
are changed by changes in environment, such as
going from warm buildings to cold weather out-
side, absorbent materials should delay the altera-
tion in heat loss. If, however, conditions are
changed by increased metabolism and resulting
sweat secretion, changes in heat loss may also be
delayed by absorbent clothing. Thus, full cooling
by evaporation might not be realized for some
time after initiation of sweating, but cooling might
continue after sweat secretion had stopped. It
would be expected that this cooling would con-
tribute to after-exercise chill.

661. ‘Local or initial’ potential of the crusta-
cean single motor axon. Ernest B. WRIGHT
AND WILLIAM J. ADELMAN.* Dept. of Physiology,
Univ. of Rochester School of Medicine and Den-
tistry, Rochester, N. Y. !

The ‘local or initial’ potentials of different lob-
ster motor axon types has been studied. With long
duration d.c. pulses it has been found that the
local response of the highly repetitive firing fiber,
opener, is of long duration, over 15 msec., and low
voltage, less than 2 mv; whereas for the non-re-
petitive firing fiber, giant fiber of ventral nerve
cord, the local response is of short duration, less
than 5 msec. and considerable voltage, over 5 mv.
With loss of repetitive firing ability, either by
deterioration or subjection to abnormal media (ex-

204

cess K or low Na concentrations), local response of
this fiber type is shortened to appear similar to the
normal local response of the non-repetitive type
fiber. Local responses of both fiber types are mark-
edly increased in duration and amplitude by low
temperatures but are decreased in duration while
increased in amplitude with high temperatures.
The duration of the local response of any fiber is
determined by the remarkable property of this re-
sponse to ‘turn off.’ This ‘turn off,’ if exceedingly
abrupt, may be followed by a period of some re-
fractoriness. This is because the abrupt ‘turn off’
is due to the development of a very small spurious
spike. The smaller the first of two local potentials,
the greater the summation of the second with de-
creasing interval between d.c. stimuli. These re-
sults suggest the release of a chemical substance
producing this ‘local or initial’ potential.

662. Comparison of effective stroke volume
and simultaneously recorded left ventricu-
lar, aortic and radial arterial pressure
pulses in aortic stenosis in man. J. LEO
Wricut (introduced by Howarp B. BurcHELL).
Mayo Fndn., Rochester, Minn.

Cardiac output (Fick) and simultaneously re-
corded left ventricular, aortic and radial arterial
pressure pulses were compared in 7 patients with
aortic stenosis who were studied by combined
catheterization of the right and left sides of the
heart and the sorta. Effective stroke volumes
ranged from 27 to 77 ml, while simultaneously
measured systolic pressure gradients across the
aortic valve ranged from 24 to 131 mm Hg and
were associated with sharply peaked left ventricu-
lar pressure pulses. The calculated area of the
aortic valves (Gorlin) ranged from 0.3 to 1.2 cm?.
The aortic and radial pressure pulses showed the
usual abnormalities in aortic stenosis, namely
prolonged build-up to the systolic maximum, an
abnormal anacrotic pause and, in contrast to the
normal, similarity between the central and radial
contours, associated with absence of amplification
of systolic pressure at the radial artery. A close
relationship between the degree of abnormality of
the pressure pulses and the magnitude of the pres-
sure gradient or the calculated valvular areas was
not apparent. Larger stroke volumes, however,
tended to be associated with more prolonged
build-up times and accentuation of the abnormal
anacrotic pause seen on the aortic pressure pulse.

663. Decreased growth of fruit fly larvae dur-
ing and after exposure to high g fields.
Cuartes C. Wunpber (introduced by CHARLES
J. Ita). Dept. of Physiology, State Univ. of Iowa,
Towa City.

Exponential growth rates for Drosophila melano-
gaster larvae were compared before, during and

FEDERATION PROCEEDINGS

Volume 15

after centrifugation for 24 hr? at fields as high ag
5000 g. It has already been demonstrated that dur.
ing exposure growth decreases with field (Prog,
Soc. Exper. Biol. & Med. 89: 544, 1955). Although
the animals are able to return to more normal
growth after exposure, there is a delayed effect in
that many are unable to emerge from the pupae,
The degree to which centrifugation affects growth
is apparently dependent upon both the stage of
development and the temperature. The principal
observations made were of physical volume; stud-
ies of the more subtle biological changes have not
yet been performed.

664. Effect of cholinesterase on superior cer-
vical ganglion of the cat. Liuoyp R. Yonce
(introduced by CHar Es R. BrassFIELD). Univ,
of Michigan Med. School, Ann Arbor.

The preganglionic nerve to the superior cervical
ganglion (SCG) of a cat (pentobarbital sodium,
40 mg/kg, i.p.) was sectioned proximally and
placed in a reversible fluid bridge electrode con-
nected to a stimulator having strength, duration
and frequency controls. The nictitating mem-
brane, attached to an isotonic lever scribing ona
kymograph, was used to indicate the level of ac-
tivity of the SCG. Stimulating the preganglionic
nerve with a constant strength and a low fre-
quency (less than 20/sec.) caused a contraction of
the nictitating membrane that rises to a maximum
and remains for the brief period of stimulation
(10-20 sec.). High frequency (more than 20/sec.)
caused a decrease in the maximum contraction and
also a subsequent exponential decrease from the
maximum even though the stimulus continued.
The contraction of the nictitating membrane ap-
pears to be a function of the rate of liberation, the
rate of hydrolysis, and the rate of synthesis of
acetylcholine. High frequencies caused a libera-
tion and destruction of acetylcholine at a greater
rate than it could be resynthesized, thus causing
a decline of the maximum contraction. Cholines-
terase (Cutter Laboratory), applied topically or
injected through the circulation of the SCG caused
a decrease in the height of contraction, a greater
effect occurring when the cholinesterase was ap-
plied topically. The higher the frequency of stimu-
lation the greater was the effect of cholinesterase,
either topically or arterially. The greater cholines-
terase effect at higher frequencies was probably
the combined action of two effects, the increased
rate of hydrolysis and the decreased amount of
liberated acetylcholine. Evidence is presented
which tests the validity of some SCG phenomena.

665. An estimation of magnitude of Paco,
Paco, differences due to unequal ventila-
tion-perfusion ratios between lobes. A. C.
Youna, C. J. Martin* anp J. Koter.* Dept. of

lume 15

high ag
1at dur-
| (Proe,
lthough
normal
ffect: in
pupae,
growth
tage of
rincipal
e; stud-
ave not

or Cer.
Yonce
). Univ,

cervical
sodium,
ly and
de con-
uration
y mem-
ng ona
1 of ac-
iglionic
ow fre-
ction of
1ximum
ulation
20/sec.)
ion and
‘om the
tinued,
ane ap-
ion, the
1esis of
libera-
greater
causing
10lines-
ally or
caused
greater
vas ap-
' stimu-
sterase,
10lines-
-obably
creased
punt of
ssented
omena.

Paco,
ontila-
ALG
ept. of

March 1956

Physiology and Biophysics, Univ. of Washington
School of Medicine, and Firland Sanatorium,
Seattle.

It has been pointed out that variations of the
ventilation-perfusion ratio in different regions of
the lungs can cause a difference between the par-
tial pressure of expired alveolar CO: and arterial
blood COz. Lobar catheter measurements of lobar
yentilation and lobar Oz consumption are used to
make a calculation of the magnitude of this differ-
ence. This calculated difference will be low since
it assumes uniformity of V/Q ratio throughout the
individual lobes. A formula for the effect of varia-
tions of the ventilation-perfusion ratio shows that
the effects are proportional to the square of the
deviations from the average. It is apparent that
deviations of r; from r in any direction cause an
increase in K. With subject in the upright position
the magnitude of K using upper and lower lobes
as the compartments is at least as high as 0.12.
(The values of r;/r may be as low as 0.4 or as high
as 1.8.) The added calculated dead space when
blood values are used is KT. If these very large
variations in r;/r persist in exercise, then it is not
unreasonable that these r,/r variations! may be the
main cause of the apparent added dead space when
blood values are used. (Aided by a grant from the
Natl. Tuberculosis Assoc.)

666. Simultaneous determination of sedi-
mentation and _ diffusion coefficients
through biological assay. Davin A. YPHAN-
TIs AND Davip F. Waveu (introduced by J. Y.
Lettvin). Massachusetis Inst. of Technology,
Cambridge.

A separation cell has been constructed which
allows a solute distribution to be established un-
disturbed during the course of an ultracentrifuga-
tion. On deceleration the cell contents are divided
along a plane of separation into predetermined
centripetal and centrifugal volumes. These may
be removed separately and assayed. The value of
Q, the fraction of the original activity remaining
in the centripetal volume, under given experimen-
tal conditions, can be accounted for by an infinite
set of paired values of S and D. Thus two runs of
sufficiently different duration yield two S vs D
curves whose point of intersection defines unique
values. If the separation plane lies in the plateau
region of uniform concentration, i.e. for large
values of ¢ = w*S/D or for small values of rt =
4w*st, the values of Q are uninfluenced by diffusion
and thus lead only to an unique value of S. This
method for determining S and S and D has been
applied to vitamin By, serum albumin, DPN,
ACTH, thrombin and other materials.

667. New bioassay for urinary LH. M. X. Zar-
row, E.S. E. Harez* anp G. Pincus. Worcester
Fndn. for Exptl. Biology, Shrewsbury, Mass.

AMERICAN PHYSIOLOGICAL SOCIETY

205

A new bioassay for the determination of LH in
the urine has been devised. The assay is based on
the ability of LH to induce ovulation in the imma-
ture rat previously treated with PMS and is a
modification of the technique involving the induc-
tion of ovulation in the adult rat hypophysectom-
ized during proestrus (Hertz, R., personal com-
munication). The following technique is used in
the present procedure. Twenty-one-day-old rats
are injected subcutaneously with 30 1u of PMS and
this is followed 56 hr. later by the subcutaneous
injection of the ovulating hormone (RowLanps,
I. W. J. Endocrinol. 3: 384, 1944). The fallopian
tubes are excised 20 hr. after the last injection and
examined for ova. If present, the ova are removed
with a fine pipette, treated with hyaluronidase and
counted. Experiments with human chorionic gona-
dotropin (HCG) indicate that dose-response
curves can be established on the basis of percent-
age of rats ovulating or number of ova released at
a certain dosage. The latter would appear to be
the end point of choice and can detect approxi-
mately 1 ru of HCG. The number of ova released
following injection with LH or HCG appears to
depend on both the priming dose of PMS and the
ovulating dose of HCG or LH. (Aided-in-part by a
grant from the Josiah Macy, Jr. Fndn.)

668. Gastrointestinal secretion and absorp-
tion of 3-methylaminoisocamphane hydro-
chloride (mecamylamine). EuGENE J. Zawot-
SKI, JoHN E. Barr, Ler W. BRAUNSCHWEIG, SUE
F, PauLtson AND AUDREY SHERMER (introduced
by Kart H. Bryer). Pharmacology Section,
Sharp & Dohme, Div. of Merck & Co., Inc., West
Point, Pa.

The ganglionic blocking agent, mecamylamine,
was administered intravenously to Heidenhain
pouch dogs at 3.05 mg/kg. In one hour two un-
stimulated dogs secreted an average of 3.7 y/ml of
mecamylamine. Two dogs administered sodium
acetate intravenously secreted 72.3 y/ml. Two
dogs given histamine intravenously secreted 25.6
y/ml, and two dogs administered metacholine in-
travenously secreted 22.0 y/ml. When 7.32 mg/kg
of mecamylamine was administered orally to two
dogs during histamine venoclysis, an average of
27.2 y/ml of mecamylamine was secreted in two
hours. Little, if any, mecamylamine was absorbed
by gastric pouch mucosa. When 24.4 ~-24.4 mg was
instilled into the pouches of four dogs, recoveries
at 15 min. averaged 78.1% while none of the com-
pound was found in plasma. Intestinal absorption
was studied in acute pentobarbital-anesthetized
dogs. Eight loops were tied off in each dog and
aliquots of mecamylamine were injected into six,
two being excised immediately, two after 15 min.,
two after 30 min. and the two controls last. Aver-
age recoveries were 87.0% for the loops immedi-

206

ately excised, 47.9% for the 15-minute sections
and 32.6% for the 30-minute tissues. Plasma levels
obtained during these experiments, averaging 2.8
mg/l]. at 15 min. and 2.7 mg/I. at 30 min., further
demonstrate the absorption of mecamylamine by
intestinal mucosa.

669. Stimulation of arterial phosphatide syn-
thesis in rabbit atheromatosis. D. B. Z11-
VERSMIT AND E. L. McCann gss.* Univ. of Ten-
nessee, Memphis.

Rabbits maintained for 5 mo. on a diet supple-
mented with cholesterol and fat showed gross ac-
cumulations of phosphatides in the thoracic aorta.
The aortic lecithin and sphingomyelin concentra-
tions increased 190 and 310% above control values,
respectively, whereas the cephalin concentration
was changed very little. Thus, sphingomyelin con-
stitutes more than 30% of the total phosphatide
of the Atheromatous aorta. Studies with radioac-
tive phosphate indicated that the incorporation of
P32 into sphingomyelin of the whole thoracic aorta
of the cholesterol-fed rabbit was more than 20
times that of the control animals. Aortic lecithin
synthesis also appeared to be greatly increased
after cholesterol feeding. Similar changes in the
turnover rate of plasma phosphatides were ob-
served. The turnover of the liver phosphatides was
affected least of all by the cholesterol regimen.
Comparison of the specific activities of individual
phosphatides in aorta and plasma confirms pre-
vious findings (Am. J. Physiol. 181: 527, 1955) that
in rabbits the phosphatides of the arterial lesion
are not derived from the plasma but are synthe-
sized in the aorta. (Supported by the Life Insur-
ance Med. Research Fund.)

670. Metabolism of mice bearing ACTH-se-
creting tumors. CLAIRE ZOMZELY,* KENNETH
H. SHuti* anp J. Mayer. Dept. of Nutrition,
Harvard School of Public Health, Boston, Mass.
In a Previous communication (Mayer, ZoMZELY

AND Furtu. Science. In press), the evolution of

ACTH-secreting pituitary tumors and their effect

on body composition and energy balance were de-

scribed: the mice bearing these tumors (‘ATO
mice’) are in positive energy balance (slightly hy-
perphagic and less active) and, in terms of com-
position, are ‘obese’ (several times as much fat)
even when not ‘overweight.’ Their body and blood
cholesterol are also high. When fatty acids and
cholesterol synthesis were studied using acetate-1-

C* in fed and fasted animals, it was found that the

ATO animals incorporated significantly (P < 0.01)

more labeled acetate into fatty acids than either

tumor-bearing adrenalectomized mice or normal
controls. In fed animals, there was no difference
as regards acetate incorporation into cholesterol.

But fasted ATO mice had a significantly (P <

FEDERATION PROCEEDINGS

Volume 1§

0.01) greater incorporation; in fact, in these ani-
mals, fasting did not decrease cholesterogenesig,
As regards carbohydrate metabolism, it was found
that the hepatic glucose-6-phosphatase activity/
gm of tissue in ATO mice was 50% greater (P <
0.001) than in the controls. A small but definite
increase in specific phosphorylase activity was also
observed in these animals. Finally, while under
fed conditions liver glycogen and blood glucose
were similar in ATO and in control animals, after
a 24-hr. fast the blood glucose of ATO mice was
still at the ‘fed’ level (40 mg % above the normal
fasted value) and their liver glycogen was more
than 10 times the normal fasted value.

671. Osmotic control of total body fluid vyol-
ume. GEORGE D. ZurIpEMA, NEVILLE P. CLARKE
AND Mary F. Minton (introduced by JAmgs P,
Henry). Aero Med. Lab., Wright Air Develop,
Center, Wright-Patterson Air Force Base, Ohio.
During recent years considerable interest has

been shown in the mechanism of total body fluid
regulation. There is accumulating evidence that
this mechanism is highly complex, perhaps involv-
ing receptors to sense blood volume and blood
pressure in specialized regions, as well as receptors
activated by changes in blood osmotic pressure,
Workers studying osmotic control mechanisms
(VERNEY, 1947) used trained conscious dogs to
study urine flow following intracarotid injections
of hypertonic solutions, and on the basis of these
studies built up an hypothesis of a center in the
hypothalamus which sensed the increase in 0s-
motic pressure and compensated by release of anti-
diuretic hormone. The following experiments were
carried out under light chloralose anesthesia in
order to eliminate the role of conditioning in alter-
ing urine flow. Intracarotid injection of normal
saline did not alter urine production, while hyper-
tonic solutions of equal osmolarity of urea, saline,
glucose, sodium sulfate and sucrose were effective
in producing antidiuretic responses. These hyper-
tonic solutions raised the osmotic pressure of caro-
tid blood by approximately 50% over a period of
10 sec. Experiments within physiological limits,
e.g., raising carotid osmotic pressure by approxi-
mately 2% over a 40-min. period, also produced an
oliguric response when hypertonic sodium chlo-
ride, sodium sulfate or urea were used. Infusions
with normal saline elicited no changes in flow. As-
say for antidiuretic hormone present in the urine
following injections suggests that the resulting
oliguria is due to release of posterior-pituitary
antidiuretic substance, rather than a neural mech-
anism.

672. Bacterial involvement in reactions to
stress in the rat. B. W. Zwerracu, S. G. HEr-
sHEY, I. Guccrone,* I. SAPHRA* AND W. ANTO-

ume 1§

Se ani-
renesis,
s found
tivity/
r(P<
definite
yas also
» under
glucose
s, after
ice was
normal
S more

id vol-
JLARKE
.MEs P,
Develop,
>, Ohio.
est has
ly fluid
ce that
involv-
| blood
ceptors
ressure,
janisms
logs to
ections
of these
> in the
in 0s-
of anti-
ts were
esia in
n alter-
normal
hyper-
saline,
ffective
hyper-
of caro-
riod of
limits,
pproxi-
uced an
n chlo-
fusions
ow. As-
e urine
sulting
tuitary
1 mech-

ons to
3. HER-
_ ANTO-

March 1956

pot. Dept, of Pathology, New York Univ.-Belle-

vue Med. Ctr. and Helen Yeamans Levy Fndn.,

Beth Israel Hosp., New York City.

Cultures of representative tissues (liver, spleen,
kidney, skeletal muscle) and blood samples of the
normal, healthy rat showed no evidence of bac-
teria, with the exception of the intestinal tract
where the fecal contents were found to contain
Klebsiella (B. lactis), E. coli, and occasionally
paracolon or enterococci organisms. Following the
interjection of several types of graded stress, bac-
terial cultures remained negative in the case of
normal rats, but showed contamination in the
blood, liver and spleen in stress imposed subse-
quent to particular experimental regimes. Predis-
posing factors, such as diet, excess cortisone, or
nonlethal body x-radiation served to introduce
bacterial contaminants into the blood, liver and
spleen following standard episodes of stress which
in normal rats were not associated with bactere-
mia. Hemorrhagic shock carried out with rigid
aseptic precautions was not associated with bac-
teremia. However, under conventional laboratory
conditions, bacterial contamination of the blood,
liver and spleen appeared in a majority of cases
and was found to be the consequence of exogenous
infection and not the result of invasion of bacteria
from the intestinal tract. The appearance of bac-
teremia was associated with a deficient readjust-
ment to hypotension and an increased mortality
when compared with the more favorable response
to hemorrhage under aseptic experimental condi-
tions. Irreversibility to blood replacement was
induced even in the absence of bacterial involve-
ment, by a more protracted period of drastic hypo-
tension. No evidence of bacteremia in either the
blood, liver or spleen was encountered in fatal
drum shock.

AMERICAN PHYSIOLOGICAL SOCIETY

207

673. Participation of the R-E system in the
readjustment to hemorrhage and trauma.
B. W. Zweiracu, J. M. McKenna* ann W.
Antopou. Dept. of Pathology, New York Univ.-
Bellevue Med. Ctr., and Helen Yeamans Levy
Fndn., Beth Israel Hosp., New York City.

The reticulo-endothelial system has been shown
to influence the response to bacterial endotoxins,
infections, total body x-radiation and several
other forms of stress. Blockade of the R-E system
in rats by the administration of either Thorotrast
(0.3 ml of a 25% solution/100 gm), proferrin (6 mg/
100 gm), chlorophyllin (50 mg/100 gm) or a special
carbon suspension (24 mg/100 gm) made the rats
uniformly more susceptible to trauma by the No-
ble-Collip drum, 80-85% fatalities occurring with a
dose only 20-30% lethal in controls. Attempts were
made to influence the resistance to trauma ac-
quired by rats after repeated exposure to sublethal
trauma. Blockade of the R-E system was the only
one of a wide spectrum of experimental contin-
gencies found to undermine the resistance of
trained or adapted rats. The protection afforded
by pharmacologic agents likewise became in-
effective following R-E blockade. Pretreatment
with R-E blockers markedly altered the capacity
to withstand hemorrhagic hypotension, as well
as the ultimate response to blood replacement
measures. In experiments carried out under asep-
tic precautions, spontaneous uptake from the
blood reservoir (decompensatory tendency) and
48-hr. survival statistics were all below normal.
The effect on the response to hemorrhage was
a time-dependent phenomenon, the predisposing
action of R-E blocking agents being lost after
4-6 hr. Pretreatment with such agents altered the
response of the peripheral vascular system, -as
manifest by the reactions to bacterial endotoxins
and to vasoactive agents.

AMERICAN SOCIETY OF BIOLOGICAL
CHEMISTS

Forty-seventh Annual Meeting
Attantic City, New Jersey, Aprit 16-20, 1956

An asterisk * following an author’s name indicates “by invitation”

674. Erythrocyte protoporphyrin and copro-
porphyrin synthesis in vitro and inhibitory
effects of certain benzimidazoles. Lynn D.
Apport, Jr., Mary J. Dopson* anp Dona.p K.
Auvi.* Dept. of Biochemistry, Med. College of
Virginia, Richmond.

Certain alkyl-substituted benzimidazole deriva-
tives prevented incorporation of N*® from N¥-
glycine into heme by chicken erythrocytes in-
cubated in vitro. To determine whether these
compounds interfered with conversion of proto-
porphyrin to heme or affected earlier reactions
involved in porphyrin synthesis from glycine, we
determined their effect on synthesis of free eryth-
rocyte protoporphyrin and coproporphyrin by
quentitative measurement of these porphyrins
produced from glycine and from 6-aminolevulinic
acid, by chicken erythrocytes in vitro. As in pre-
vious experiments on N-heme synthesis from
N¥-glycine, benzimidazole and 2-methylbenzimi-
dazole had some inhibitory activity, but with
similar concentrations (.007 m), 5-methyl-, 2,5-
dimethyl-, 5,6-dimethyl-, and 2-ethyl-5-methyl-
benzimidazole (.003 m) completely inhibited syn-
thesis of free erythrocyte protoporphyrin and
copropofphyrin from glycine. Inhibition of proto-
porphyrin and coproporphyrin synthesis from
glycine indicated a step in the metabolic pathway
prior to porphyrin ring formation probably was
involved. In contrast, porphyrin synthesis from
6-aminolevulinic acid was not blocked by these
compounds. Inhibitory effects of these alkylben-
zimidazoles on heme and porphyrin synthesis thus
appear to be at some step prior to the formation of
é-aminolevulinic acid from glycine, hence a reac-
tion early in the metabolic pathway. This is con-
sistent with our hypothesis that these compounds
might be affecting a reaction which may be neces-
sary for other biosynthetic mechanisms, such as
virus multiplication and azo dye liver tumor for-
mation. (Aided in part by a medical student fel-
lowship of the Natl. Fndn. for Infantile Paralysis
to D. K. Auvil.)

675. Reduction of a thioketone by a model for
DPNH. Ropert H. ABELES AND FRANK H,
WESTHEIMER (introduced by Eric G. Batt).
Dept. of Chemistry, Harvard Univ., Cambridge,
Mass.

It has been shown by Westheimer, Vennesland
and collaborators (in McE.Lroy anp Guass, The
Mechanism of Enzyme Action. Baltimore: Johns
Hopkins Press, 1954, p. 357) that, in many en-
zymatic reductions involving DPNH (reduced di-
phosphopyridine nucleotide), hydrogen is trans-
ferred directly (i.e. without exchange with the
medium) from DPNH to the substrate. In order
to gain further information about the mechanism
of enzymatic reductions involving DPNH the
study of some nonenzymatic model systems was
undertaken. In the course of this investigation it
was found that DPNH can reduce thiobenzophe-
none nonenzymatically. N-benzyl-1,4-dihydro-
nicotinamide, which is structurally similar to
DPNH, proved a convenient model for the coen-
zyme. This compound reduces the thioketone to
thiobenzhydrol and is itself converted to the N-
benzyl-nicotinamide cation. The thiobenzhydrol
produced in this reaction was isolated and identi-
fied as a solid derivative. When N-benzyl-1,4-di-
hydronicotinamide-4-D was employed, deuterium
was transferred directly to the thioketone to form

(CsHs)2CDSH. The oxidation reduction reaction’

was also carried out in D.O as solvent. It has thus
been demonstrated that the reduction of a thio-
ketone in our model system proceeds as does the
enzymatic reduction of ketones, with direct hy-
drogen transfer.

676. Effect of oxaloacetate on pyruvate and
succinate oxidation by avocado particles.
M. AsBRANSKY* AND J. B. Braue (introduced by
Stoney Roserts). Dept. of Subtropical Horti-
culture, Univ. of California, Los Angeles.

When oxaloacetate was used as the sole sub-
strate for a particulate preparation from the avo-
cado fruit, no oxygen uptake could be observed

208

So - <= «ff

lel for
vK H,
BALL).
bridge,

esland
s, The
Johns
ny en-
sed di-
trans-
th the
. order
anism
H the
ns was
tion it
zophe-
hydro-
lar to
» coen-
one to
the N-
hydrol
identi-
1,4-di-
terium
o form

action *

as thus
a, thio-
yes the
set. hy-

e and
ticles.
iced by
Horti-

le sub-
he avo-
yserved

March 1956

in a Warburg manometer. However, when the re-
action mixture was later chromatographed on
paper, malate and citrate could be identified as
products of the reaction, suggesting the following
mechanism:

1. Oxaloacetate — Pyruvate + CO:

—2H :
2. Pyruvate + Oxaloacetate ———> Citrate
+ COz

2H
3. Oxaloacetate ce, Malate

Additional evidence to the postulated pathway
was: 1) oxaloacetate, when added to pyruvate,
caused a lag in the oxygen uptake proportional to
the amount of oxaloacetate added; 2) oxaloacetate
was decarboxylated by the preparation. COz evo-
lution in the presence of oxaloacetate as the sole
substrate was greatly inhibited by arsenite; 3) in
the presence of arsenite neither malate nor citrate
were produced in the reaction; 4) addition of
malonate did not eliminate the formation of mal-
ate. When oxaloacetate was added to succinate in
the reaction mixture, a similar lag period was pro-
duced. This lag, however, does not seem to be due
to an oxidation and reduction proceeding simul-
taneously, but is most likely due to a direct in-
hibition of the system. Calcium ions, when added
to the reaction mixture, increased the inhibition
considerably, while magnesium ions or ATP re-
versed it.

677. Conversion of hydroxyproline to gluta-
mate in bacterial extracts. Ev1sAH ADAMS.
Dept. of Pharmacology, New York Univ. College
of Medicine, New York City.

A soil bacterium, provisionally Pseudomonas,
was selected by growth on hydroxy-t-proline as
sole organic substrate. Resting cells adapted to
either hydroxy-u-proline or allohydroxy-p-proline
are preadapted to the other; soluble extracts of
adapted cells utilize either isomer and form L-glu-
tamate, identified chromatographically and by
enzymatic decarboxylation. The remaining iso-
mers, hydroxy-p-proline and allohydroxy-u-pro-
line, are poor substrates for whole cells or extracts
and form no detectable glutamate. L-Proline is a
non-adaptive substrate for whole cells but is not
utilized by extracts that degrade hydroxyproline.
Glutamate production corresponds to only about
half the hydroxyproline utilized; the discrepancy
is only partly explained by further degradation of
glutamate. Tests for accumulated intermediates
were negative for the following groups: pyrroline
compounds (o-aminobenzaldehyde), a-keto acids
(semicarbazide) and carbonyl compounds (2,4-
dinitrophenylhydrazine). Several specific possible
intermediates, tentatively excluded by failure to

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

209

stimulate respiration of hydroxyproline-grown
cells or glutamate formation by the corresponding
extracts, are naturally-occurring a-hydroxyglu-
tamate (VIRTANEN, Acta chem. Scand. 9: 175, 1955),
4-ketoproline (synthesized by Dr. Arthur Patch-
ett) and 2-pyrrolidone 5-carboxylic acid. The sub-
strate activity of hydroxyproline isomers indicates
the requirement for a specific configuration at
carbon 4 but not at carbon 2. Previously suggested
schemes for hydroxyproline degradation are analo-
gous to known proline-glutamate interconversions
via glutamic semialdehyde. On this basis, p-glu-
tamate (in place of the u-glutamate observed)
would be formed from allohydroxy-p-proline. In-
version at the glutamate stage is excluded by sub-
strate inactivity of p-glutamate. Remaining alter-
natives include earlier epimerization at carbon 2
of hydroxyproline or a reaction sequence not anal-
ogous to proline degradation.

678. Estrogenic activity and metabolism of
diethylstilbestrol diphosphate. Ernest C.
ADAMS, JR.,* HELEN M. FreE* anp ALFRED H.
FREE. Miles-Ames Research Lab., Elkhart, Ind.
Intravenously administered diethylstilbestrol

diphosphate has been used successfully in the

treatment of prostatic carcinoma (FLocks, J.

Urology 74: 549, 1955). The present report describes

studies of the estrogenic activity of diethylstil-

bestrol diphosphate in the rat. It also describes
studies of the metabolism of the compound when
administered intravenously to the dog. Bioassays
using the increase in uterine weight of immature
female rats as the criterion of estrogenic activity
show that diethylstilbestrol diphosphate possesses
activity comparable to that of diethylstilbestrol
after either oral or subcutaneous administration.

In the metabolism studies, diethylstilbestrol di-

phosphate was administered intravenously to

adult female dogs and urine samples were obtained

by catheterization. Chemical determination of di-

ethylstilbestrol and its conjugates in urine was

made by a colorimetric antimony pentachloride
method. Chemical and biological assays both in-
dicated that metabolites representing approxi-
mately 10% of a 100-mg dose of diethylstilbestrol
diphosphate were excreted in the urine with prac-
tically all of the activity appearing during the
first 24 hr. Fractionation of the urine by ether ex-
traction at pH 2.2 and px 8 provided a basis for
establishing the nature of the metabolites in the
urine. Very little free diethylstilbestrol was ex-
creted. The majority of the metabolite behaved
as a conjugate unlike the diphosphate. Distribu-

tion of the conjugate between ether and px 4.9

phosphate buffer indicated it was the monoglu-

curonide.

679. Electrophoretic and enzymatic hetero-
geneity of beta-galactosidase of E. coli.

210

FREDERICK ALADJEM,* JAcoB DuBNorrF, Dan H.
CAMPBELL* AND ELISABETH BARTRON* Gates
and Crellin Labs. of Chemistry and Kerckoff Labs.
K. of the Biological Sciences, California Inst. of
Technology, Pasadena.

From E. coli K-12 an enzyme preparation was
obtained which hydrolyzes lactose, but not ortho-
nitro-phenyl-beta-galactoside (ONPG). Extracts
of E. coli (buffer-alumina grinding) were subjected
to starch electrophoresis using potassium phos-
phate buffer, » = 0.1, po 7.6. Extracts from several
types of cell preparations were used: cells grown
on lactose, cells grown without lactose but adapted
to lactose for short periods of time and unadapted
cells. Enzymatic activity against lactose was
found in 3 electrophoretically separable compo-
nents in all cell extracts. Enzymatic activity
against ONPG was found in the same 3 electro-
phoretic components from extracts of cells grown
on lactose. In the other 2 preparations enzymatic
activity against ONPG was found in only 2 of the
3 electrophoretic components. The 3rd was active
against lactose but entirely inactive toward
ONPG.

680. Metabolic significance of blood amino
nitrogen levels. ANTHONY A. ALBANESE,
Louise A. Orto* AND Dorortuy N. ZAVATTARO.*
Nutritional Research Labs., St. Luke’s Hosp.,
New York City.

Recently, by means of newly developed proce-
dures we have studied relationships between fast-
ing blood amino N levels and protein nutrition in
healthy and convalescent children and adults.
The existing nutritional status was ascertained by
nitrogen balance measurements and reference to
age-height-weight tables. Averages of fasting
blood amino N levels of 22 children (5-15 yr.) and
101 adults (40-95 yr.) showed good statistical cor-
relation with the degree of malnutrition expressed
in percentage of underweightness. In most in-
stances deviations from this correlation could be
accounted for by the existence of excessive hydra-
tion or dehydration. Subsequently, we found that
blood amino N increments 1 hr. following oral
protein dosages (adults, 0.2 gm/K and children,
0.4 gm/K) provided a more accurate criterion of
protein nutritional status than fasting amino N
levels alone. In accord with the laws of diminish-
ing returns, this increment was found to be in-
versely related to the decrease of body weight, N
balance and fasting amino N from the norm.
Also, by this modality it became possible to dif-
ferentiate true from false high fasting amino N
levels which may prevail in chronic disease states.

681. Heat labile form of citrovorum factor in
human urine. A. M. ALBRECHT (introduced by
E. L. R. Stoxstap). Nutrition and Physiology

FEDERATION PROCEEDINGS

Volume 15

Section, Research Div., American Cyanamid Co.,

Lederle Labs., Pearl River, N.Y.

When 50 mg folic acid and 1 gm ascorbie acid
were administered orally to adults at least 50% of
the subsequent urinary excretion of citrovorum
factor (CF) was in the form of heat labile sub-
stance(s) as determined by aseptic assay with
Leuconostoc citrovorum (Pediococcus cerevisiae),
When such urine was chromatographed on paper in
a 0.1 m pH 54 acetate buffer system and subse-
quently bioautographed (L. citrovorum) a growth
zone at Rr 0.70+.05 corresponding to 5-formyl-
tetrahydrofolic acid (5-CHOTHFA, leucovorin)
and a growth zone at R¢ 0.35+-.05 were found. The
latter growth zone was not present in chromato-
grams from urine that had been autoclaved 30
min. and is referred to as heat labile citrovorum
factor (HLCF). HLCF could not be distinguished
by paper chromatography or paper ionophoresis
from anhydroleucovorin A or a freshly prepared
solution of 10-CHOTHFA. Although air could be
bubbled for 30 min. through urine containing
HLCF without significant loss of activity, HLCF
became very susceptible to air oxidation on purifi-
cation by large scale paper chromatography. Like
HLCF, anhydroleucovorin has slight 5-CHO-
THFA activity in conventional autoclaved assays;
but, aspetic assay of anhydroleucovorin, freshly
prepared in water or at pH 2 for several hours
markedly increases microbiological activity.

682. Histidine biosynthesis: imidazoleglyc-
erol phosphate dehydrase. Bruce N. Amgs
(introduced by J. Retp). Natl. Insts. of Health,
Bethesda, Md.

It has been proposed previously that the path-
way of histidine biosynthesis in Neurospora crassa
involves the sequence: p-erythro-imidazoleglycerol
phosphate -— imidazoleacetol phosphate —
L-histidinol phosphate. These 3 phosphate esters
are accumulated by various histidineless mutants
of Neurospora. (AMES AND MITCHELL, J. Am.
Chem. Soc. 75: 1015, 1953; J. Biol. Chem. 212: 687,
1955). It is not yet known whether in Neurospora
L-histidinol phosphate is converted to histidine
through t-histidinol, a precursor of histidine in
E. coli and yeast. (VoGEt et al. J. Am. Chem. Soc.
73: 1897, 1951; Apams, J. Biol. Chem. 209: 829,
1954). Imidazoleacetol phosphate is converted
to t-histidinol phosphate by a pyridoxal phos-
phate enzyme, imidazoleacetol phosphate trans-
aminase, which has been purified from Neurospora
and which catalyzes the reaction: imidazoleacetol
phosphate + t-glutamate = t-histidinol phos-
phate + a-ketoglutarate. (AMEs AND HoRECKER,
J. Biol. Chem. In press). Another enzyme, imid-
azoleglycerol phosphate dehydrase, has now been
purified from Neurospora. This enzyme forms
imidazoleacetol phosphate by removing the ele-

lume 16
vid Co.,

ic acid
50% of
»vorum
le sub-
y with
visiae),
yaper in
subse-
growth
formyl-
ovorin)
id. The
‘omato-
ved 30
ovorum
zuished
yhoresis
repared
ould be
taining
_HLCF
1 purifi-
iy. Like
5-CHO-
assays;
freshly
1 hours
y.

leglyc-
. AMES
Health,

e path-
a crassa
zlycerol
ate —
e esters
nutants
J. Am.
112: 687,
Lrospora
istidine
idine in
em. Soc.
09: 829,
nverted
il phos-
e trans-
wrospora
leacetol
>| phos-
RECKER,
e, imid-
ow been
e forms
the ele-

March 1956

ments of water from p-erythro-imidazoleglycerol
phosphate: ‘Im - CHOHCHOHCH,OPO;H: —
Im-CH.COCH:OPO;H:. The activity of the
enzyme is greatly increased in the presence of cys-
teine and manganese (II), while ethylenediamine-
tetraacetate is a very potent inhibitor. The puri-
fication and properties of the dehydrase will be
discussed. The dehydrase and transaminase,
which interconvert the 3 phosphate esters, are
not active on the corresponding dephosphorylated
imidazoles, which are also accumulated by the
histidineless mutants studied. The occurrence of
these enzymes therefore further supports the
proposed sequence in histidine biosynthesis
involving these phosphate esters.

683. Electrophoretic pattern of the serum
protein of mice of a new leukemia strain.
Marie A. ANDERSCH AND FRANK H. J. FIGGE.
Div. of Clinical Pathology, Dept. of Medicine
and the Dept. of Anatomy, Univ. of Maryland
Med. School, Baltimore.

The serum protein patterns, obtained by paper
electrophoresis, of 24, C;H/Fg strain leukemic
mice were compared with the patterns of 15 non-
leukemic mice of the same strain and with those
of 13 cancer resistant, Cs; black mice maintained
in the same laboratory. Mice of the C;H/Fg
strain exhibit a 55% incidence of lymphatic leu-
kemia in our colony although Law has observed
only a 30% incidence. The mean values for total
protein, albumin, alpha and beta globulins of the
leukemic mice were significantly lower than those
fractions in the nonleukemic mice of the same
strain. The gamma globulin and the alpha globulin
of the nonleukemic mice were lower than the
corresponding fractions in the serum of the mice
of the cancer resistant strain.

684. An enzymatic defect in a congenital hu-
man disease, galactosemia. E. P. ANDERSON,*
Kurt J. IsseELBACHER* AND HERMAN M. Kat-
cKAR. Natl. Insts. of Health, Bethesda, Md.
Studies on the metabolism of galactose-1-phos-

phate (Gal-1-P) in tissues have indicated the

importance of the enzyme termed ‘PGal uridyl
transferase’, which catalyzes the exchange of
a-Gal-1-P with uridinediphospho-glucose, forming
a-glucose-1-phosphate and _ uridine-diphospho-
galactose. This enzyme has been partially purified
from calf liver and the mechanism and character-
istics of the reaction investigated. Assays for the
reaction have been developed for use in crude or
purified cell-free systems, and it has been found
that Gal-1-P can be utilized by this mechanism
in liver systems, but apparently not in extracts
from brain or mammary gland. Specific studies on
human erythrocytes have shown the PGal trans-
ferase reaction to be consistently present and

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

211

highly active in blood of normal persons, but to
be lacking in blood in the hereditary (A. Houzeu
AND G. M. Komrower. Arch. Dis. Childhood
30: 155, 1955) disease, galactosemia. The defect
in galactose metabolism manifest in this disorder
could be explained on this basis, as could the
findings of Schwartz et. al. (Biochem. J. 59: xxii,
1955) on the accumulation of Gal-1-P in erythro-
cytes of galactosemic children. The block would
appear to be in the biosynthesis of the transferase
itself since experiments on a lack of a cofactor or
the existence of an inhibitor of the enzyme in
galactosemic blood have so far been negative.

685. Structural and functional relationships
in ribonuclease. CHrRIsTIAN B. ANFINSEN.
Natl. Heart Inst., Bethesda, Md.

Earlier studies have suggested that the en-
zymatic activity of ribonuclease may be depend-
ent upon only a restricted portion of its single
cross-linked chain. Thus, extensive rupture of
hydrogen bonds by urea and various limited pro-
teolytic cleavages do not lead to inactivation.
(e.g. Ricuarps, C. rend Carlsberg, Ser. Chim.,
29: #19, 329, 1955). On the other hand cleavage
of what appears to be a single peptide bond by
pepsin completely inactivates. This process in-
volves the release from the C-terminal end of the
chain of the tetrapeptide asp-ala-ser-val. The rate
of release is quantitatively correlated with in-
activation. It has been found that a change in
spectral properties, characteristic of the rupture
of hydrogen bonds between tyrosine-hydroxy] and
carboxylate groups of the sort described by
Crammer and Neuberger, can also be demon-
strated during the course of this specific inactiva-
tion. A very rapid change of this sort is quanti-
tatively related to the rate of tetrapeptide release
and subsequent further changes in the extinction
at 278 mu occur more slowly. The rupture of a
hydrogen bond, involving the beta-carboxyl group
of the aspartic acid present in the tetrapeptide
released during the fast reaction, is compatible
with molecular models of the C-terminal portion
of the chain. A consideration of the above observa-
tions, together with current studies on the nature
and distribution of the disulfide bridges of ribo-
nuclease, have suggested that the ‘active center’
of the protein is associated with a constellation
of residues near the C-terminal end of the mole-
cule.

686. Distribution of infused estrogens in
human plasma. H. N. ANTONIADEs, F. INGER-
soLL, J. W. McArtuur AND R. B. PENNELL
(introduced by F. R. N. Gurp). Protein Fndn.
Labs. and Vincent Memorial Hosp., Boston,
Mass.

The present study concerns the distribution of

212

infused estrogens in plasma fractions. Two male
volunteers were injected intravenously, for this
purpose, each with 100 mg of estrone-sulfate;
2 other volunteers were each injected with 100 mg
of free estrone. Phlebotomy followed 2 hr. after
estrogen administration, 500 ml of whole blood
being collected in each instance through cationic
exchange resin (Dowex 50). The separated plasma
from each volunteer was fractionated immediately
using Cohn’s cold-ethanol methods 6 and 9. The
fractions obtained were I, III-O, II + IIIw,
IV-1, IV-4, V, and VI. They were assayed bio-
logically for estrogens by a modification of the
method of Bulbring and Burn. In both experi-
ments following the injection of estrone-sulfate,
the estrogenic activity was found associated with
fraction V which is essentially albumin. Activity
recovered in fraction IV-1 was due to the small
amounts of albumin present in this fraction.
Specific precipitation of this albumin from frac-
tion IV-1 with antihuman albumin horse serum
left the supernatant inactive, while the estrogenic
activity was recovered in the albumin precipitate
Fraction III-O containing the 8-lipoproteins was
inactive. When free estrone was injected, the
activity was found mainly in fraction IV-1.
Immuno-precipitation of the albumin from this
fraction removed only part of the activity, while
the main estrogenic activity remained in the
supernatant a-globulins. Fraction III-O was again
free of estrogenic activity. Fraction VI (super-
natant) in all experiments was active possibly due
to the presence of free estrogens.

687. Inhibition of the growth of Leuconostoc
mesenteroides P-60 by L-penicillamine and
its reversal by serine. H. VASKEN APOSHIAN
AND JAMES A. Seruirr (introduced by J. P.
Lampooy). Pharmacology Dept., Vanderbilt
Univ. School of Medicine, Nashville, Tenn.
Using the antimetabolite approach in a rational

search@for serine antagonists, wL-penicillamine
(a-amino-8-mercapto-8-methyl butyric acid) and
also DL-a-amino-$-mercapto butyric acid, isomer
A, were found to inhibit the 72-hr lactic acid
production of Leuconostoc mesenteroides P-60
under the conditions for the microbiological assay
of serine. By increasing the concentration of
serine, the inhibition of growth, measured by
lactic acid production, caused by t-penicillamine
was reversed. DL-a-amino-8-mercapto butyric
acid, isomer A, is a more potent growth inhibitor
than L-penicillamine.

688. Dietary protein and carotene utilization.
Lotte ARNRICH AND DorotHy J. PEDERSON
(introduced by AaNEs Fay Morgan). Dept. of
Home Economics, Univ. of California, Berkeley.
The utilization of small doses of an aqueous

FEDERATION PROCEEDINGS

Volume 16

carotene suspension as measured by liver and
kidney vitamin A deposits was affected by the
level of dietary proteins. Young male rats, pre-
viously depleted of their vitamin A stores were fed
200 meg of carotene daily for 4 wk. Those receiving
11% dietary casein had 8 mcg of vitamin A in
their liver. Those receiving 22% casein had 32 meg
of liver vitamin A and those receiving 40% casein
had 60 meg of liver vitamin A. Kidney deposits of
the vitamin were similarly affected. When similar
groups of rats fed 22 and 40% casein were re-
stricted in their caloric intake to match the food
consumption of those given the low protein ration,
the resulting liver vitamin A deposits were almost
twice as large as those found in corresponding
groups fed ad libitum. Feeding preformed vitamin
A to similarly prepared rats gave results quite
different from those obtained by the use of caro-
tene. Rats on the low protein diet stored as much
total vitamin A in their liver as those fed 40%
casein, while the concentration per gram of tissue
was doubled in the more slowly growing animals on
11% dietary casein. Since the effect of protein
intake could be demonstrated on feeding carotene
but not preformed vitamin A, it seems likely that
the underlying mechanism involves a step in the
absorption or conversion of carotene rather than
in the utilization of the vitamin A formed by the
processes.

689. Biogenesis of the pyridine ring in higher
plants. S. Aronorr. Inst. for Atomic Research
and Dept. of Botany, Iowa State College, Ames.
Reciprocal grafts have shown that in Nicotiana

species, the ultimate synthesis of nicotine occurs

in the roots, while that of its isomer, anabasine,
occurs both in roots and leaves (Dawson, Am,

J. Bot. 31: 351-355, 1944). We have confirmed the

last biochemically by partial degradation of

anabasine from C'“O.-photosynthesizing excised
leaves of N. glauca. Chromatographically-isolated
anabasine, converted to the dipicrate, was shown
to have the same specific activity (per carbon of
anabasine) as the nicotinic acid derived from it.

Degradation of the nicotinic acid produced C0:

and pyridine of identical specific activity (per

carbon). Consequently it is highly probable that
the entire molecule is uniformly labeled and there-
fore of simultaneous origin. Feeding of lysine-2-

C™“ or hydroxylysine-e-C'* to excised leaves

resulted in only insignificant conversion to anaba-

sine. Feeding of 3-hydroxyanthranilic acid-car-
boxyl-C"4 to excised pea leaves plus stems did not
result in detectable radiotrigonelline, although
this system converts nicotinic acid rapidly to
trigonelline. It is concluded that the major path-
way of pyridine ring biogenesis in higher plants

is at present unknown. (Supported in part by a

grant from the National Science Fndn. We ac-

— se ak an Oe a ee ae ak eee Se a ee

— @2 @.

ace°206dUC~«CSlUC

ume 1§

r and
Dy the
3, pre-
ere fed
eiving
1 A in
32 meg
casein
sits of
similar
are Te-
ie food
ration,
almost
onding
itamin
: quite
f caro-
3 much

nals on
protein
.rotene
ly that
in the
or than
by the

higher
esearch
Ames.
cotiana
occurs
basine,
N, Am.
ned the
ion of
excised
solated
. shown
rbon of
rom it.
d C0,
ty (per
le that
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ysine-2-
leaves
anaba-
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> plants
rt by &
We ac-

March 1956

knowledge with gratitude a sample of hydroxy-
lysine-e-C™ from Dr. F. M. Sinex.)

690. Lipide choline and fatty acid oxidation
in rat liver preparations. CAMILLO ARTOM.
Dept. of Biochemistry, Bowman-Gray School of
Medicine, Winston-Salem, N. C.

The lipide composition of the liver and the
oxidation of long-chain C*-fatty acids by liver
homogenates were studied in rats receiving either
choline, or compounds which may interfere with
the synthesis of choline or of choline-containing
phospholipides. In rats maintained on low-protein
diets with added guanidoacetic acid, choline (in
the diet, or by injection shortly before the animals
were killed) raised the lecithin level and en-
hanced the oxidation of fatty acids in vitro. Similar
effects were observed by supplementing the diets
with triethylcholine. Addition of diethanolamine
to choline-deficient diets caused more marked
decreases in the amounts of lipide choline. How-
ever, the rates of fatty acid oxidation in these
livers were as high as in those of rats receiving
choline. After injection of pt-ethionine, the livers
of female rats on a stock diet exhibited high
lecithin levels, increased fat contents, and a
markedly reduced ability of the isolated tissue to
oxidize fatty acids. pxi-methionine prevented
completely both the fatty infiltration and the
decrease in the rate of fatty acid oxidation. Ad-
ministration of choline along with ethionine was
ineffective. The significance of these findings will
be discussed in relation to the hypothesis that
the enhancement of the oxidation of fatty acids
in vitro by choline, administered in vivo, may be
due to an increased formation of liver lecithins.
(Aided by U.S. Atomic Energy Commission,
Contract No. AT-(40-1)-1638.)

691. Pathways of glucose metabolism in
diabetic liver. James AsHMoRE,* GrEorGE F.
CanHILL, JR.* anp A. Barrp Hastines. Harvard
Med. School, Boston, Mass.

The utilization of glucose by all mammalian
tissue requires that this hexose be first phos-
phorylated to glucose-6-phosphate. In liver,
glucose-6-phosphate may 1) be converted to
glycogen; 2) be glycolyzed via the Embden-
Meyerhof pathway; 3) be metabolized via the
phosphogluconate oxidation pathway, or 4) be
hydrolyzed to glucose and inorganic phosphate.
An attempt has been made to evaluate the relative
tates of these 4 pathways using liver slices from
fed normal and alloxan diabetic rats incubated in
a medium of intracellular cationic composition.
With glucose-1-C™“, glucose-6-C™“ and fructose-
U-C™ as added substrates, the incorporation of
C“ into glucose and glycogen and the oxidation
to C“O. were measured. In cases in which glucose-

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

213

1-C" served as the substrate the maximum number
of molecules of glucose-6-phosphate metabolized
via the phosphogluconate oxidation pathway
would be equal to the number of moles of C“O,
produced since carbon 1 of glucose is lost as CO,
via this route. Assuming that all of the glucose
disappearing from the incubation medium is first
converted to glucose-6-phosphate, and that the
concentration of intracellular glucose-6-phosphate
is negligible it has been calculated that in liver
from normal rats 100 molecules of glucose-6-
phosphate would be distributed as follows: 18 to
glycogen, 4 via the phosphogluconate oxidation
pathway, 56 to glucose, and the remainder (20)
via the Embden-Meyerhof pathway. In liver
slices from alloxan diabetic rats the distribution
is: 2 to glycogen, 13 via the phosphogluconate
oxidation pathway, 75 to glucose, and the re-
mainder (5) via the Embden-Meyerhof pathway.

692. DNA produced by E. coli after T2 bacteri-
ophage infection in the presence of pro-
flavin. L. AsTRAcHAN (introduced by Wa.po
E. Coun). Biology Div., Oak Ridge Natl. Lab.,
Oak Ridge, Tenn.

It has been reported (R. I. De Mars, Virology
1: 83, 1955) that HF. colt infected with bacteri-
ophage in the presence of proflavin does not form
viable phage but does synthesize the major
chemical constituents of phage; that is, phage-like
DNA (hydroxymethyleytosine DNA) and phage-
like protein (morphological and immunological
identity). To test the possibility that DNA formed
in the proflavin system may differ in nucleotide
sequential arrangement from normal phage DNA,
the polynucleotides produced by enzymic degrada-
tions were compared. The proflavin system DNA
was uniformly labeled with P*? and then mixed
with 15-20 times its amount of authentic phage
DNA. The total DNA was then isolated and
hydrolyzed with either DNAse or whole snake
venom (diesterase plus monoesterase). Both
enzyme systems produced a high yield of poly-
nucleotide fragments (dinucleotide or greater).
The hydrolyzates were chromatographed on
Dowex-1 or cellulose-triethanolamine columns.
In each chromatographed fraction, the chemically
determined phosphate was predominantly a
measure of material from authentic phage DNA,
whereas radioactivity measured only the con-
tribution of proflavin system DNA. Any differ-
ences in specific activities of the products would
reflect sequential differences in the intact DNA’s.
Comparisons of specific activities showed the
2 DNA materials to be the same with respect to
content of various polynucleotides produced by
DNAse. The polynucleotides produced by the
snake venom enzymes also had the same specific
activities with the exception of 1 fraction which

214

had a lower specific activity, indicating greater
contribution from authentic phage DNA.

693. Inhibition of prostatic acid phosphatase
by a-hydroxyl carboxylic acids. BERNARD
AXELROD AND Grace E. SEWELL.* Dept. of
Biochemistry, Purdue Univ., West Lafayette, Ind.
The observation by Abul-Fadl and King (Bio-

chem. J. 45: 51, 1949) that L-tartaric acid but not
its enantiomorph is an inhibitor of prostatic acid
phosphatase, but not of phosphatases in general,
is of particular interest in connection with eluci-
dating the nature of the active enzymic site. A
number of a-hydroxy! carboxylic acids have been
tested in order to define the structural features
requisite for such inhibition. It appears that an
a-hydroxy] group in the p-configuration is needed
in addition to the carboxyl group. It also appears
that the 8-carbon must be either a carboxyl carbon
or have attached to it one of the following groups:
carboxyl, hydroxyl or carboxymethylene. The
most potent inhibitor after L-tartaric acid was
p-glyceric acid. p-phosphoglyceric acid, which
was very slowly split by the enzyme, was not
inhibitory. The mono- and di-ethyl esters of tar-
taric acid, carefully purified to exclude free
tartaric acid, were mildly inhibitory. They were
not deesterified by the enzyme. The inhibition
by .L-tartaric acid was truly competitive when
tested against a number of diverse substrates,
approximately the same value for K; always
being obtained. A possible mechanism of inhibi-
tion will be discussed.

694. Role of adenosine triphosphate in the
enzymatic activation of carbon dioxide.
B. K. BacuHawat,* J. F. WoEssNER, JR.* AND
M. J. Coon. Dept. of Biological Chemistry, Med.
School, Univ. of Michigan, Ann Arbor.

In the course of earlier studies on the enzymatic
carboxylation of 6-hydroxyisovaleryl coenzyme
A (HIV CoA), it became apparent that 2 distinct
steps might be involved: 1) CO2 + ATP @ active
CO. + pyrophosphate; and 2) active CO. +
HIV CoA = £-hydroxy-6-methylglutaryl coen-
zyme A (HMG CoA). In support of this proposed
reaction sequence, the enzymes catalyzing the
individual reactions have been separated. A 60-
fold purified preparation of the CO:-activating
enzyme from heart tissue liberates pyrophosphate
from ATP (Reaction 1), but does not accomplish
the carboxylation of HIV CoA (Reaction 2) unless
a preparation of hydroxyisovaleryl carboxylase is
also added. The carboxylase used in such experi-
ments has been shown to be free of any detectable
amount of the CO:-activating enzyme. It was
suggested earlier that active CO. might have a
‘high energy’ bond linking carbonate to the phos-
phate group of adenylate and permitting car-

FEDERATION PROCEEDINGS

Volume 1§

boxylation of the relatively inert methyl groups
of HIV CoA (Federation Proc., 14: 762, 1955. A
compound believed to be adenosine phosphory|
carbonate, obtained as one of the products of the
reaction of ethyl chloroformate with the silver
salt of adenylic acid, has been shown to be capable
of carboxylating HIV CoA in the presence of the
carboxylase fraction alone. These and other find-
ings suggest that active CO: is closely related to
or identical with adenosine phosphoryl] carbonate,

695. Utilization of the purine moiety of ri-
bosides and purines. M. Earu Batis anp
Dorris J. Hutcuison.* Sloan-Kettering Inst.
for Cancer Research, New York City.

The incorporation of the purine moiety of
adenine, adenosine and adenylic acid into the
nucleic acid of a series of strains of Streptococcus
faecalis has been determined. The organisms used
were a parent and several mutants resistant to
certain anti-metabolites. The purine of adenosine-
5’-phosphate was not used at all, although the
free base and the riboside serve as sources of both
of the purines of the nucleic acid of these several
strains of the organism. Some of these organisms
were able to use the adenine moiety of adenosine-
3’-phosphate as a source of nucleic acid adenine
but none converted it to nucleic acid guanine,
This observation contrasts with that made with
Lactobacillus casei which organism is capable of
using the adenine of adenosine-3’-phosphate for
the synthesis of both nucleic acid purines. Pre-
vious studies with bacteria have shown that the
phosphate of nucleotides is not incorporated with
the purine. It follows then that the ribotides are
not used per se. However, since the adenine moiety
of adenosine is converted to guanine, adenylic
acid cannot be used via adenosine or free adenine.
It also follows that, if adenylic acid is an inter-
mediate in the conversion of exogenously supplied
adenine to nucleic acid, adenine is incorporated
via a pathway independent of that of purine
interconversion.

696. Tricarboxylic acid cycle in mammalian
carcinoma cell. STANLEY BARBAN AND H. 0.
ScuuLzE (introduced by E. A. PETERsoN).
Natl. Microbiological Inst., Natl. Insts. of Health,
Bethesda, Md.

Cell suspensions of a mammalian carcinoma cell
(strain HeLa) exhibit a high rate of oxidation.
Cell-free extracts prepared by sonic disintegra-
tion were found to contain all the enzymes of the
tricarboxylic acid cycle. Among the reactions
carried out by these preparations were citrate or
isocitrate to a-ketoglutarate; a-ketoglutarate to
succinate; succinate to fumarate and malate;
malate to oxalacetate and pyruvate; and citrate
synthesis via the condensing enzyme. In addition,

ae ee ae ae

"re Boe woe co f+ . ©

ume 1§

groups
955. A
phoryl
of the
silver
apable
of the
sr find-
ited to
onate,

of ri-
[iS AND
g Inst,

ety of
ito the
ococcus
ns used
tant to
nosine-
igh the
of both
several
ranisms
nosine-
adenine
uanine,
Je with
able of
ate for
18. Pre-
hat the
ed with
ides are
‘moiety
denylic
denine.
n inter-
upplied
porated

purine

malian
> H. 0.
ERSON).
Health,

oma cell
idation.
integra-
3 of the
eactions
trate or
arate to
malate;
| citrate
ddition,

March 1956

a soluble e-ketoglutaric acid oxidase was demon-
strated in ‘these extracts, the cofactor require-
ments of which were shown to be coenzyme A,
Mg** and diphosphopyridine nucleotide. Both
t-malic dehydrogenase and the ‘malic enzyme’
were also found in the HeLa cell.

697. Path of glutamate decomposition by
Clostridium tetanomorphum. H. A. BARKER,
J. T. Wacusman,* A. MuncH-PETERSEN* AND
M. A. Witson.* Dept. of Plant Biochemistry,
Univ. of California, Berkeley.

Glutamate is fermented to acetate, butyrate,
carbon dioxide and ammonia by C. tetanomor-
phum. Balance and tracer experiments have shown
that the fermentation does not involve the TCA
eycle. Cell-free extracts convert glutamate to
pyruvate, acetate, a-ketoglutarate and ammonia.
The last 2 products are formed by a TPN specific
glutamic dehydrogenase. Under some conditions,
mesaconate (2-methyl fumerate) accumulates as
a product of glutamate breakdown. Mesaconate
is converted to pyruvate and acetate at a rate
sufficient to indicate that it may be an interme-
diate in glutamate fermentation.

698. Liver coenzyme A in hypophysectomized
immature rats. Paut D. BartLett, PAMELA
GRIMMETT* AND SHIRLEY SHELATA.* Edsel B.
Ford Inst. for Med. Research, Detroit, Mich.
The observation that liver coenzyme A is not

significantly increased in hypophysectomized

immature rats stimulated with growth hormone
presents an anomaly in an otherwise direct rela-
tionship between liver coenzyme A concentration

and the growth process (Federation Proc. 14:

177, 1955). Results of recent studies appear to

indicate that this deviation is due neither to a

suboptimal dietary intake of certain precursors

required for the biosynthesis of coenzyme A

during a greatly stimulated growth process, nor

to the fact that the hypophysectomized immature
rat is essentially hypothyroid. Thus, for example,
livers obtained from ad libitum fed hypophysec-
tomized immature rats, maintained on stock diet
containing either 1% u-cystine or 2% pu-methio-
nine for a period of 20 days, and from similar
groups of rats stimulated with growth hormone
during the final 10-day period of amino acid sup-
plementation, were found to contain, respectively,
141+23.6, 1836428, 131+11.3 and 12343 Kaplan-

Lipmann units coenzyme A per gram wet weight.

Supplementation of ad libitum fed hypophysec-

tomized immature rats with daily intraperitoneal

injections of 200 ug calcium pantothenate for a

period of 20 days and, in addition, of a similar

group of rats with growth hormone during the
final 10-day period, resulted in livers which
assayed, respectively, 153415 and 142+24.5 Kap-

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

215

lan-Lipmann units coenzyme A per gram wet
weight. Stimulation of ad libitum fed hypophys-
ectomized immature rats with both growth hor-
mone and thyroxine for a period of 10 days pro-
duced no significant alteration in liver coenzyme
A concentration.

699. Soluble DPNH and succinic dehydro-
genases from the electron transfer particle.
R. E. Basrorp AND B. pE BERNARD (introduced
by J. W. WiuutaMs). Enzyme Inst., Univ. of
Wisconsin, Madison.

The electron transfer particle (ETP) of beef
heart mitochondria (cf. abstract of J. L. Glenn
and F. L. Crane) catalyzes the oxidation of suc-
cinate and DPNH by oxygen. The 2 primary
dehydrogenases can be extracted from the particle
and solubilized by the following procedures:
1) When water suspensions of ETP are acidified
to pH 4.2 in the presence of 9% alcohol, warmed
to 43° (H. EpetnHocn et al., J. Biol. Chem., 197:
97, 1952) and then centrifuged, the supernatant
contains a soluble flavoprotein (I) with DPNH
dehydrogenase activity (30 um DPNH/mg/min.
at 38° with ferricyanide as acceptor, lum/mg/min.
with cytochrome c as acceptor). The increase in
specific activity is about 10-fold over that of
ETP. To prepare a soluble succinic dehydro-
genase, ETP is first fragmented to the succinic
dehydrogenase complex (D. E. Green et al.,
J. Biol. Chem., 217: 551, 1955) which in turn is
converted to an acetone powder. A soluble suc-
cinic dehydrogenase, II, can be extracted from
the powder by a modification of the method of
Singer and Kearney (Biochim. Biophys. Acta,
15: 151, 1954). The acetone powder is first ex-
tracted with 0.1 m phosphate buffer, px 7 followed
by an extraction with 0.06 m Tris buffer px 8.9.
The second extract is further purified by absorp-
tion and elution from tri calcium phosphate gel.
The specific activity of the soluble dehydrogenase
is 2-3 times that of SDC and 5 times that of ETP
as measured with either ferricyanide or phenazine
methosulfate as electron acceptor. II has as its
prosthetic groups, flavin (probably FAD) and
inorganic iron as has been previously shown by
Singer and Kearney.

700. Oxidation of sulfide by an adaptive sys-
tem in rat liver. CLAUDE F. Baxter,* Ropert
Van REEN* AND Paut B. Pearson. McCollum-
Pratt Inst., Johns Hopkins Univ., Baltimore, Md.
Extracts of perfused rat liver homogenates

contain a heat labile (I) and a heat stable (II)

system, which catalyze the oxidation of sulfide to

thiosulfate. The thiosulfate end product is identi-
fied enzymatically by reacting with cyanide in
the presence of rhodanese to form thiocyanate.

Both systems require atmospheric oxygen and

216

are at least partially soluble (Spinco supernatant).
They are essentially nondializable. System I has
a& pH optimum (7.2-7.4), temperature optimum
(35-50°C) and shows substrate saturation. Within
the ranges tested, system II has neither a pH nor
temperature optimum and does not become sub-
strate saturated. Most of the sulfide which dis-
appears during the reaction can be accounted for
by the thiosulfate formed. The addition of chelat-
ing agents to extracts stimulates the rate of
thiosulfate formation. At concentrations of 10-‘m,
the effect of 8-hydroxyquinoline is predominantly
on I, while the stimulation by versene is more
pronounced in II. Complexes of cobalt with ver-
sene, crystalline bovalbumin and other com-
pounds, show ‘sulfide oxidase’ activity and may
serve as model systems. This requirement for co-
baltous ions is fairly specific. A 2- to 9-fold
increase in the sulfide oxidase level (per mg of
protein) is observed when rats are fasted for
24 hr. This is in agreement with the elevated thio-
sulfate levels which are observed in the urine of
fasting animals (FRoMAGEOT AND Royer, Enzy-
mologia 11: 361, 1944). A possible interrelationship
between the sulfide oxidase systems and cysteine
desulfhydrase is being studied.

701. Biochemical differences in the effects of
growth hormone and of insulin adminis-
tration. GrorcE H. Beaton anp Doris M.
Curry (introduced by E. W. McHenry).
Dept. of Public Health Nutrition, Univ. of
Toronto, Toronto, Canada.

The administration of suitable doses of either
growth hormone or of insulin to plateaued female
rats caused equal body weight increases. Growth
hormone-treated animals showed an increase in
total body protein and a decrease in total body
fat; insulin treatment caused an increase in total
body fat and no marked increase in protein.
Growth hormone produced an increase in liver
weight¢ the livers of these rats had decreased
activities of 2 transaminases. Insulin did not cause
these liver changes. Blood urea was decreased and
blood amino nitrogen increased by growth hor-
mone but not by insulin. The changes in body
weight and composition consequent to insulin
treatment are explicable solely on the basis of the
increased food intake. Growth hormone causes
metabolic alterations which induce the utilization
of body stores of fat to provide energy so that
protein may be spared and retained.

702. Bacterial dismutation of 5-methyltryp-
tophan. ERNEstT BEERSTECHER, JR. AND
ELEANoR J. Epmonps.* Univ. of Texas, Dental
Branch, Houston.

Previous studies (J. Biol. Chem. 192: 497, 1951)
have shown that Escherichia coli is capable of

FEDERATION PROCEEDINGS

Volume 16

breaking down 5-methyltryptophan to form an
indole, but that indole is essential in catalytic
amounts to initiate the reaction. Studies of the
rate of the reaction have now demonstrated that
during the course of the process increased amounts
of tryptophan are formed. Using differential
chemical and microbiological assay methods which
measure 5-methyltryptophan, tryptophan, 5-meth-
ylindole and indole separately, it has been
found that the 5-methyltryptophan does not
serve as a substrate for tryptophanase. Indole
apparently serves as a co-factor by which Esche-
richia coli first converts the methyltryptophan to
tryptophan. The latter is then cleaved to indole
and pyruvate in the conventional tryptophanase
reaction.

703. Tumor lipogenesis in vitro. R. W. Brae
AnD J. A. TrEw.* Dept. of Med. Research, Univ.
of Western Ontario, London, Canada.

Based on the work of Medes et al. (Cancer
Research 13: 27, 1953) generalizations are being
made that tumors have a low capacity for lipo-
genesis. We have measured the rate of incorpora-
tion of acetate-1-C™ into the lipids of 4 trans-
plantable rat tumors, and compared the rate with
that of liver, kidney, spleen, lung and intestinal
mucosa. Under the conditions of our experiments
in vitro the tumors have as great or greater a
capacity for the incorporation of acetate into
lipids as have the most active normal tissues,
(Aided by a grant from the Natl. Cancer Inst. of
Canada.)

704. Effects of 8-sitosterol on development
and regression of cholesterol atherosclero-
sis in rabbits. W. T. Breuer, W. L. AntHony*
AND G. D. Baxer.* Edsel B. Ford Inst. for Med.
Research, Henry Ford Hosp., Detroit, Mich.
Preliminary experiments have shown that £-sit-

osterol is effective in reducing the rate of serum

cholesterol build-up in rabbits maintained on a

high cholesterol diet. To study further the effec-

tiveness of B-sitosterol, 30 female albino rabbits
were maintained on a stock diet supplemented
with 3% corn oil and 1% cholesterol to develop
atherosclerosis. At the end of 3 months, 10 rabbits
were killed. The aortic arch and 5-7 em of aorta,
samples of liver and blood were removed. The
remaining 20 rabbits were divided into 2 groups
for regression study. For a 4-month period, group!

received the stock diet plus 3% corn oil; group II

received the stock diet plus 3% corn oil and 2.5%

B-sitosterol. Periodically serum cholesterol was

determined. At the end of 4 months, the rabbits

were killed and samples of aorta, liver and blood
removed for evaluation. The aortas were stained,
and the area of plaque involvement determined.

Then total lipid and cholesterol were assayed.

Sse = = = - oo ce oo eS lO

| — a — a,

ume 16

rm an
talytic
of the
d that
nounts
rential
; which
-meth-
been
28 not
Indole
Esche-
han to
indole
hanase

. Brae
, Univ.

‘Cancer
> being
yr lipo-
orpora-

trans-
te with
-estinal
riments
eater &
te into
tissues.
Inst. of

pment
sclero-
THONY*
or Med.
ich.

at, B-sit-
f serum
d on a
ie effec-
rabbits
mented
develop
rabbits
f aorta,
od. The
- groups
group!
yroup IT
nd 2.5%
rol was
rabbits
d blood
stained,
rmined.
assayed.

March 1956

The samples of liver were used for cholesterol
determination. The data obtained from the deter-
minations showed: 1) @-sitosterol slightly in-
creased the rate of serum cholesterol regression;
2) after development of cholesterol atheroscerosis,
there was very little aorta plaque regression in
either of the groups—f-sitosterol reated or con-
trol; 3) during the development of atherosclerosis,
liver cholesterol reached very high levels, which
regressed to near control values in 4 months.
g-sitosterol treated animals regressed slightly
more than the untreated animals.

105. Influence of serum inhibitor on anti-
phlogistic action of trypsin. J. Morton
Be1Ler,* Rosert R. BRENDEL* AND GUSTAV
J. Martin. Research Labs., The Natl. Drug Co.,
Philadelphia, Pa.

This report deals with studies undertaken to
determine the role of serum inhibitor in the anti-
phlogistic action of trypsin. Sherry et. al, (J. Lab.
& Clin. Med. 40: 942, 1952) observed incomplete
neutralization of trypsin by large excesses of
trypsin inhibitor in vivo. They reported that in
undiluted plasma combination between trypsin
and trypsin inhibitor was not stoichiometric
(J. Clin. Investigation 33: 1308, 1954). Nord et al.
(Nature 176: 789, 1955) suggested that trypsin
derivatives having less combining power with the
serum inhibitor might be clinically superior.
With edema produced by egg-whites in rats as a
test system, it was found that the antiphlogistic
effect of trypsin could be inhibited by soybean
and ovomucoid inhibitor, but only at concentra-
tions 5-10 times above those producing inhibition
of tryptic activity, in vitro. The antiphlogistic
action of a number of trypsin derivatives (sup-
plied by Dr. F. F. Nord), as measured by mini-
mum effective dosage, bore no relation to their
dissociation constants with serum trypsin in-
hibitor. It appeared rather to be a function of
proteolytic activity. Chronic administration of
trypsin to guinea pigs caused only small increases
in serum trypsin inhibitor levels. Results indicate
that combination with serum inhibitor does not
influence the antiphlogistic action of trypsin.

706. Spectra of intermediate oxidation stages
of flavins. H. Bemnert, J. G. Hauce* anp
E. Pacs.* Enzyme Inst., Univ. of Wisconsin,
Madison.

When 1 of the fatty acyl CoA dehydrogenases
is reduced by substrate, a broad absorption band
(Amax 560 mu) appears. A band of the same char-
acteristics and intensity has now been observed
during the few seconds in which reduction of these
flavoproteins by dithionite proceeds or during
subsequent reoxidation by oxygen. We conclude
that the band indicates an intermediate oxidation

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

217

stage of the prosthetic flavin, which is stabilized
in presence of substrate. Kinetic and equilibrium
studies by spectrophotometry suggest the follow-
ing interactions (S, substrate; F, flavoprotein;
SH.2, FH, reduced forms): SH, + F S=H:—F =
SH—FH = S—FH. = S + FH:. SH—FH is
thought to be the form indicated by the absorp-
tion band. Intermediate oxidation stages of ribo-
flavin (e.g., KuHN, WaGNER-JAuUREGG, Ber., 67:
361, 1934; Micuar.ts et al., J. Biol. Chem. 116:
587, 1936) and of a flavoprotein (Haas, Biochem.
Z. 290: 291, 1937) have been described previously.
We have recorded spectra of intermediate oxida-
tion stages of FAD. The intermediate at px 7.4
and 4.3 shows a band (Amax 580 my) very similar
to that of the enzyme, whereas this band is missing
at pH 9.8 and 12.2. At pH 0.5 the maximum is at
490 mz. The intermediates of FAD but not of
the flavoprotein have broad absorption bands in
the near infrared: Amax 850; 830; 1040; 1040; 830 my
at pu 0.5; 4.38; 7.4; 9.8; 12.2, respectively. The
relative ratio of intensities of infrared to visible
bands varies with px. If one assumes the same
molar extinction coefficient for the intermediate
stage of free and bound FAD at px 7.4, then its
concentration is about 20 times greater for en-
zyme-bound FAD.

707. Peptidase activity of swine kidney. FRep
E. Betu (introduced by EvANGELINE Papa-
GrorGE). Dept. of Biochemistry, Emory Univ.,
Emory University, Ga.

A preparation having peptidase activity toward
L-leucinamide, glycylglycine and glycyl-u-leucine
has been obtained from a pancreatin-protease
digest of homogenized swine kidney. The prepara-
tion involved digestion for 2-4 wk., alcohol frac-
tionation followed by shaking of the active frac-
tion with octanol-chloroform, treatment first
with Norit and then with Amberlite IR4-B, and
finally dialysis. Little or no loss of total activity
resulted in any of these steps, all of which were
conducted at room temperature and at a px of
approximately 8. The complete procedure brought
about a minimal 300-fold increase in the pro-
teolytic coefficients, calculated on the basis of
total nitrogen. At px 7 a 280/260 absorption ratio
of 0.7 is observed with the dialyzed material.
This material is active toward the above sub-
strates in the absence of added metal ions. 0.001 m
Mn* or 0.004m Mgt increases the activity
toward tu-leucinamide, whereas 0.001 m Cott
moderately inhibits hydrolysis of this substrate
but increases the activity toward glycylglycine.
These ions do not appear to influence the activity
toward glycyl-t-leucine. The dialyzed material
also contains all the cysteinylglycinase activity
of the pancreatin-protease digest. Some success
has been achieved in the separation of the leucine

218

aminopeptidase and cysteinylglycinase activities.
Preliminary studies have been made which sug-
gest that the dialyzed solution also contains
alkaline phosphatases.

708. Fat emulsions. Effects on serum proteins.
Rutu R. BeENeERITO, KATHERINE Formusa,
W.S. SINGLETON AND J. L. WuiTE (introduced
by A. M. Autscnut). U.S.D.A., Southern
Regional Research Lab., New Orleans, La.

As part of a study of fat emulsions for intra-
venous alimentations, work was done on the
interaction of fat emulsions with serum proteins.
The mobilities and relative percentages of protein
fractions and lipids in pooled normal human
serum, dog serum, rat serum and in serum-emul-
sion mixtures were determined by means of paper
electrophoresis at pH 8.6. The influence of the
addition of various fat emulsions to normal human
serum in vitro, and to dog serum in vitro and in
vivo has been investigated. All emulsions selected
contained 15% by weight of cottonseed oil, 5%
dextrose, but different emulsifiers. In most of the
serum-emulsion mixtures, the mobility of the
fat was the same as that of the naturally occurring
fat in normal serum. With emulsions containing
soybean phosphatides as emulsifiers, the fat mo-
bility was greater. In these latter mixtures, the
a fraction was notably absent, whereas in the
former mixtures the a fraction was essentially
unaltered. Anomalous electrophoretic patterns
produced in the presence of certain emulsifiers
yield a possible explanation of the adverse effects
encountered in some instances when certain in-
travenous fat emulsions are administered.

709. Heterogeneity of serum albumin. REIN-
HOLD BENESCH AND RutH E. BEenescu.* Inst.
for Enzyme Research, Univ. of Wisconsin,
Madison.

It was shown previously that crystalline bovine
serum albumin (BSA) is inhomogeneous with
respect to its —SH group; } of an —SH group
per mole reacts rapidly with Ag* in neutral
aqueous solution, while 3 reacts only in the
presence of high concentrations of urea. It has
now been found that: 1) this phenomenon is not
due to any alteration of the protein during its
preparation, since the same ratio of reactive to
unreactive —SH groups (2:1) is found in crystal-
lized BSA, Cohn’s fraction V, and even in whole
serum, human or bovine. 2) Sodium dodecyl
sulfate can be used instead of urea to liberate the
unreactive —SH groups. 3) The urea denaturation
is reversible upon dilution below px 6, but is
irreversible in neutral or alkaline solution. Thus
BSA exposed to 8 m urea at pH 6 or below and then
diluted to 0.8 mM urea shows a titration of 3 of
an —SH group per mole, whereas the value of a

FEDERATION PROCEEDINGS

Volume ig

sample denatured at pH 7.4 or above and simi-
larly diluted, approximates to one —SH group
per mole. This is probably due to the occurrence
(in the presence of 8 M urea) of an exchange reae-
tion between the mercaptide ion and the disulfide
bonds of the protein. At pH values below 6 this
reaction does not occur, since the concentration
of mercaptide ion is too small. 4) Disulfide inter.
change can be prevented by silver ions as well ag
by protons. Thus a solution of serum albumin at
pH 7.4 shows a titration of 1 mole of —SH per
mole of protein in 8 m urea. If, however, such a
solution is treated with 1 mole of Agt per mole
of protein and then diluted 10-fold, only } of an
—SH group per mole is found in the diluted solu-
tion. This demonstrates that 34 of a mole of silver
which was bound by the protein in 8 M urea is
actually displaced again when the molecule re-
turns to its ‘native’ configuration.

710. Renewal of acid-soluble purines and
nucleic acid purines in tumor-bearing mice
studied with sodium formate-C". L. L,
BENNETT, JR.* AND Howarp E. SKIPPER,
Kettering-Meyer Lab., Southern Research Inst.,
Birmingham, Ala. (Affiliated with Sloan-Ketter-
ing Inst.).

The specific activities of acid-soluble purines
and DNA and RNA purines of Sarcoma 180,
intestine, spleen, kidney and carcass of the mouse
have been compared at periods of 1-120 hr. fol-
lowing a single injection of C'*-formate. In all
tissues the acid-soluble purines had reached their
maximal specific activity at 1 hr.; the DNA and
RNA purines reached maximal specific activity
at 6-12 hr. The specific activity of the total acid-
soluble hypoxanthine was higher than those of
the acid-soluble adenine or guanine or nuclei¢
acid adenine or guanine throughout the experi-
ment, suggesting ‘de novo’ synthesis of a hypoxan-
thine-containing metabolite in mammalian cells.
Within a period (1-96 hr.) during which the tumor
increased in mass 6-fold there was no significant
loss of C4 from the tumor DNA or RNA purines.
As a result of either turnover of polynucleotides
or cellular expulsion, certain rapidly dividing
tissues (intestine and spleen) lost considerable
polynucleotide purine activity within a period of
24 hr. after maximal labeling.

71l. Amide-N of glutamine as source of
guanine amino group. MARIAN BENTLEY*
AND RicHARD ABRAMS. Inst. of Research, Monte-
fiore Hosp., Pittsburgh, Pa.

We have shown that soluble extracts of rabbit
bone marrow catalyze the amination of xanthosine
phosphate (XMP) to guanosine phosphate when
ATP, Mg*', and u-glutamine or L-glutamate are
added to the extract (J. Am. Chem. Soc. 77: 4179,

del

cor

the

lume 1§

d simi-

group
urrence
re Teac.
isulfide
6 this
tration
e inter-
well ag
min at
SH per
such a
sr mole
2 of an
d solu-
f silver
urea is
ule re-

Ss and
gz mice
L. L.
<IPPER,
v Inst.,
-Ketter-

purines
na 180,
» mouse
hr. fol-

In all
-d their
NA and
ictivity
al acid-
hose of
nucleic
experi-
/poxan-
n cells,
> tumor
nificant
yurines.
eotides
ividing
derable
riod of

rce of
NTLEY*
Monte-

rabbit
thosine
e when
ate are
7: 4179,

March 1956

1955). This,reaction was observed independently
by Lagerkvist in pigeon liver extracts (Acta
Chim. Scand. 9: 1028, 1955). We have found that
azaserine, if present in excess over the amino
donor, partially inhibited the amination of XMP,
suggesting that glutamine was the true N-donor.
To further confirm this hypothesis, glutamine
containing 30.6 atom % excess N" in the amide-N
was used to aminate XMP-C”. Calculations
based upon isotope assays on the guanine isolated
from the reaction mixture indicated that the
amino group of the guanine formed from XMP
contained 28.1 atom % excess N'. Thus only
the amide-N was utilized. That the action of
glutamate in promoting the reaction results from
its conversion to glutamine was shown by com-
paring N?°-glutamate plus NH,Cl with glutamate
plus N'H,Cl. Only in the latter case was N™
incorporated into guanine even though guanine
was formed from XMP in both experiments.
(Supported by a grant-in-aid from the American
Cancer Society and by Atomic Energy Commis-
sion contract AT (30-1)-1818.)

712. Mechanism of action of aconitase from
Aspergillus terreus. RONALD BENTLEY AND
Ciara P. Turessen.* Dept. of Biochemistry
and Nutrition, Grad. School of Public Health,
Univ. of Pittsburgh, Pittsburgh, Pa.

In a previous communication (Science 122:
330, 1955) it was shown that the final step in
itaconic acid biosynthesis by A. terreus (NRRL
1960) is decarboxylation of cis-aconitic acid by
cis-aconitic decarboxylase (CAD). The cell-free
CAD preparations contain an aconitase since
CO: and itaconic acid are also formed from p-iso-
citrate and citrate. The rate of CO. formation
from p-isocitrate is about one half that from
cis-aconitate, and 2 to 3 times that from citrate;
these ratios vary with the preparation. The aconi-
tase is inhibited competitively by L-isocitrate.
Since incorporation of the complete succinate
molecule into itaconate produced in intact cul-
tures is inconsistent with the known tricarboxylic
acid cycle reactions (Communications, 3rd Inter-
national Congress of Biochemistry: p. 41, 1955),
the mechanism of action of the mold aconitase
was investigated. Incubation of CAD-aconitase
preparations with asymmetrically labeled citrate
(containing C™ in the carboxyl groups originally
derived from oxaloacetate) produces CO. which
is only slightly radioactive, whereas the itaconate
formed has the same specific radioactivity as the
citrate. Assuming the CAD removes the secondary
carboxyl group of cis-aconitate it follows that
dehydration of citrate by the mold aconitase
takes place at the bond originally formed by
condensing enzyme. The mechanism of action of
the mold aconitase apparently differs, therefore,

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

219

from that of mammalian aconitase. This observa-
tion explains the incorporation of the complete
succinate molecule into itaconate and agrees with
the earlier result of Corzo and Tatum on incor-
poration of asymmetrically labeled citrate using
an intact culture (Federation Proc. 12: 470, 1953).

713. Adenyl-acylates in the activation of
acyl groups. Paut Bere anp Greoraia NeEw-
ton.* Dept. of Microbiology, Washington Univ.
School of Medicine, St. Louis, Mo.

The over-all reaction of fatty acid activation
in yeast, bacteria and animal tissue, now well
established in the literature, is as follows: 1)
ATP (A5PPP) + RCOOH + CoA-SH = RCOS-
CoA + A5P + PP. We have described the puri-
fication of an enzyme from yeast (J. Am. Chem.
Soc. 77: 3163, 1955) which converts acetate to
acetyl CoA in the following manner: 2) ATP +
acetate — adenyl-acetate + PP; 3) Adenyl-ace-
tate + CoA = acetyl CoA + A5P. Further evi-
dence supporting this formulation has been
obtained. With ATP, acetate, hydroxylamine
and the enzyme there was a formation of 0.71 um
of acethydroxamic acid, 0.76 um of PP, and 0.73 um
of A5P. In the absence of acetate, 0.06 um of
acethydroxamic acid, 0.10 um of PP and 0.07 um
of A5P were found. A5P-C™ exchanges with ATP
only in the presence of both acetate and CoA. In
the presence of ATP, A5P-C", acetate and CoA,
0.24 um of A5P was incorporated into ATP, where-
as when either acetate or CoA was omitted less
than 0.008 um was incorporated. Acetate-C™
does not exchange with the acetyl moiety of
acetyl CoA unless A5P and PP are present. Under
conditions where 25-30% exchange occurred with
A5P and PP present, less than 1% exchange
occurred when either of these were removed. A
reaction analogous to (2), utilizing L-methionine
in place of acetate, has been studied with an en-
zyme isolated from yeast. Other amino acids do
not replace methionine. With ATP, methionine,
hydroxylamine and enzyme there is a net forma-
tion of A5P, PP and methionine hydroxamic acid.
Further work with other amino acid-catalyzed
PP-ATP exchanges and attempts to synthesize
adenyl-amino acids is in progress.

714. Micromethod for the determination of
acetylable steroids from blood and urine.
Davin L. BEeRiINER* (introduced by ALEJAN-
DRO ZAFFARONI). Dept. of Anatomy, Univ. of
Utah College of Medicine, Salt Lake City.

A procedure has been developed for the deter-
mination and identification of minute amounts
of steroids. The blood or urine samples were ex-
tracted with chloroform before and after hydrol-
ysis with beta-glucuronidase. The chloroform
extracts were then purified and dried under

220

nitrogen prior to acetylation with acetic anhy-
dride-1-C'* (S.A. 2mc/mm) and pyridine. The
mixture was chromatographed on activated silica
gel. The C-19 radioactive acetate steroids were
separated from the radioactive corticosteroid
acetates using various mixtures of benzene,
chloroform and methanol through the column.
The fractions were further purified by partition
paper chromatography according to the procedure
of Zaffaroni: The C-19 acetates were separable in
the hexane-formamide system and the corticos-
teroid acetates in the benzene-formamide system.
Various regions of radioactivity were found and
identified. A comparable experiment was done
using known radioactive C!‘ ring labeled testos-
terone, corticosterone, cortisone, cortisol and
tetrahydrocortisone, and a definite pattern of
separable zones of radioactivity was obtained.
The recoveries were quantitative. This method
can determine and identify 0.1 gamma of an
acetylable steroid. (Supported by PHS Grant
C-2349 (CS) PET.)

715. Fractionation of the lipid stain, Sudan
Black B. Epwarp W. Brermgs, JR.* anp HuGH
J. McDonaup. Dept. of Biochemistry, Grad.
School and Stritch School of Medicine, Loyola
Univ., Chicago, Ill.

In an attempt to quantitate the method of pre-
staining lipoproteins before separating them by
means of ionography, a study was initiated on
the lipid stain, Sudan Black B. (Federation Proc.
14: 733, 1955). It has been shown that different
dye lots vary considerably in their fat-staining
ability. It was decided to fractionate the dye in
an attempt to obtain a homogeneous highly
specific lipid stain. If this compound were made
available it would provide a quantitative reagent
for total lipid determination as well as for absolute
amounts of alpha and beta lipoproteins as com-
pared tp relative amounts now being determined.
This would also facilitate the attainment of re-
producible results from one laboratory to another,
a condition which does not now obtain. A chro-
matographic column 40 cm in height was wet-
packed with a celite-silicic acid mixture to a
height of 10 cm. A reservoir kept the solvent,
‘commercial isooctanes’, at a height of 25 cm
above the packing and the flow rate uniform at
80 ml/hr. After 96 hr. 4 fractions had been col-
lected. At this point acetone was added to the
system (3% by volume) and this composition
was maintained for the rest of the run. After
26 hr., during which time 5 more fractions had
come off, the column was dried and an immobile
fraction eluted off. The visual fractions (first in
the list being first from the column) were: yellow,
red, orange, green, blue, greenish blue, blue, blue,
blue and black, respectively. All the blue frac-

FEDERATION PROCEEDINGS

Volume 1§

tions ranged in absorption maxima from 580 my
to 600 mu. Blue fraction 9 became colorless at px
2.4 and below. Qualitative tests indicate that all
10 fractions stain to some degree. Lipids stained
with orange fraction 3 fluoresced under ultra
violet light. Work is being carried out to find the
best and most specific fat stain and to prepare
the compound in the pure state.

716. Reaction of human serum £-lipoglobulin
with macromolecular polysulfate esters,
Peter BERNFELD AND JEROME 8S, NISSELBAUM.*
Cancer Research and Cancer Control Unit, Tufts
Univ. School of Medicine, Boston, Mass.

In the range of pH 7.5-8.6, B-lipoglobulin hag
been found to be the only human serum protein
capable of interacting with macromolecular
polysulfate esters. As the result of this interaction,
the formation of either a soluble or an insoluble
lipoglobulin-polyanion complex can be observed,
depending on the chemical nature of the macro-
molecular sulfate ester. An insoluble lipoglobulin-
polyanion complex is formed if the polyanion
does not possess functional groups other than
—OS;H and —OH. Thus, the polysulfate esters
of amylopectin, amylose, dextran and cellulose,
containing 1 to 3 sulfate groups per glucose unit,
and polyvinyl sulfate with 0.9 sulfate groups per
repeating unit precipitate §8-lipoglobulin. A
polyanion with a branched structure (sulfated
amylopectin) yields a less soluble complex with
B-lipoglobulin than a chemically related, un-
branched polyanion (sulfated amylose). Among
many polyanions tested, sulfated amylopectin
gives the most insoluble complex with £-lipo-
globulin. If, in addition, the polyanion contains
carboxyl groups (sulfated carboxymethyl cellu-
lose, sulfated pectic acid), acetylamino, amino or
sulfamate groups (chitin and chitosan sulfuric
esters), or combinations of them (heparin, sul-
fated hyaluronate or sulfated chondroitin sulfate),
soluble complexes with 6-lipoglobulin are obtained
which are characterized by their electrophoretie
behavior. The influence of sulfate content and
molecular weight of the polyanion on the forma-
tion and solubility of the complex has been
examined. Fibrinogen also forms complexes with
the above named polyanions. The fibrinogen-
polyanion complexes are more soluble than those
with 6-lipoglobulin.

717. Distribution of some constituents of
hens’ egg yolk between its soluble and its
particulate fraction. M. J. BessMAn*, GER-
HARD ScuminptT, M. D. Hicxrey* anv §. J.
THANNHAUSER. Dept. of Biochemistry and The
Boston Dispensary, Tufts Univ. Med. School,
Boston, Mass.

Despite the fact that the particulate fraction

PS Se a ee eee ee ee eee

ip s a. a2 a2 = 2 eS ee eee

ful
71!

a a, ee

ali

lume 1§

580 my
S at pa
that all
stained
r ultra
ind the
prepare

obulin
esters,
BAUM.*
, Tufts

lin hag
protein
lecular
‘action,
soluble
served,
macro-
obulin-
lyanion
r than
> esters
liulose,
se unit,
ups per
lin. A
ulfated
2x with
d, un-
Among
opectin
B-lipo-
ontains
| cellu-
nino or
sulfuric
in, sul-
ulfate),
btained
horeti¢
nt and
forma-
s been
es with
inogen-
n those

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und its
‘, GER-

nd The
School,

raction

March 1956

of egg yolk (yolk granules) has attracted the
attention of investigators since Miescher and
Hoppe-Seyler, no reliable information is avail-
able concerning the distribution of the constitu-
ents of yolk between its soluble phase and the
granules because separation of both phases is
difficult owing to the high viscosity of egg yolk.
This difficulty can not be overcome by dilution
owing to alteration of solubility properties of
yolk constituents. Since it was found possible to
achieve sedimentation of the yolk granules by a
12-hr. centrifugation of undiluted egg yolk at
approximately 20,000 g, it was of interest to study
the distribution of some yolk constituents be-
tween the soluble and insoluble phase under these
conditions, avoiding solubility alterations. The
granules contained the total amounts of phospho-
protein, and 85% of the iron and the calcium. In
contrast to the current assumption, 90% of the
yolk phospholipides are not associated with phos-
phoprotein, but are present in the supernatant
yolk plasma, possibly in lipoprotein combination
with phosphorus-free proteins. The neutral fats
are practically exclusively present in the super-
natant yolk plasma. The proteins of the granules
consist only to maximally one-third of phosvitin.

718. A water-soluble synthetic diet. SANroRD
M. Brrnspaum, Mitton Winitz,* Rosin H
Brooks* AND JESSE P. GREENSTEIN. Lab. of
Biochemistry, Natl. Cancer Inst., Natl. Inst. of
Health, Bethesda, Md.

A completely water-soluble synthetic diet has
been prepared and fed to rats in 50% solution
from calibrated drinking tubes. The diet is com-
posed entirely of chemically defined constituents:
optically pure L-amino acids as the source of
essential as well as of nonessential nitrogen,
organically-bound phosphate, crystalline vita-
mins, glucose and a water-soluble salt mixture.
The clear solution, which required a separate
supplement of essential fats and fat soluble vita-
mins, supports the growth of weanling animals
through maturity at a rate which is at least
equivalent to that of similar strain animals raised
on a standard laboratory chow diet. No obvious
symptoms of any deficiency are observed during
this period. Preliminary findings indicate that the
reproductive and lactating capabilities of rats
raised on this diet from the weanling stage are
normal. The requirement for L-arginine for opti-
mal growth on the synthetic diet can be partially
fulfilled by p-arginine in lieu of the L-isomer.

719. Urinary output of adrenaline in normal
human males. Fritz Bischorr AND CHARLES
L. Gray.* Santa Barbara Cottage Hosp. Research
Inst., Santa Barbara, Calif.

A modification of the Lund fluorimetric adren-

aline method (Acta pharmacol. 5: 231, 1949, 6:

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

221

137, 1950) and the Shaw isolation procedure
(Biochem. J. 32: 19, 1988) was used. Because of
the controversial status of noradrenaline deter-
mination, adrenaline was oxidized at px 3, at
which noradrenaline reacts but slightly (within
experimental error). No attempt was made to
determine or correct for noradrenaline. It would
require more than 6 times as much noradrenaline
as adrenaline to introduce an error of 10% in our
adrenaline determination. The urine samples
were acidified with citric acid to a pH at which
adrenaline was shown to be stable. The stability
of urinary adrenaline at pH 7.5-8.0 as reported
by Mann (J. Pharm. & Pharmacol. 12: 1024, 1953)
was not confirmed. The rate of adrenaline excre-
tion in 7 normal males ranging in age from 25 to
55 yr. was 0.9 (low), 7.6 (high) and 2.3 (mean)
gamma per hour collected during the entire wak-
ing period and 0.8 (low), 4.3 (high) and 1.8 (mean)
gamma per hour collected during the entire sleep-
ing period. These values are higher than those
reported by von Euler and Lundberg (J. Appl.
Physiol. 6: 551, 1954); our low values falling in
the range of their high values. A possible explana-
tion is our technique of collection to prevent
urinary adrenaline destruction. Our values are
not corrected for the destruction of adrenaline
in the urine while in the bladder. This may be
considerable at neutral or alkaline pus. (Sup-
ported by the Carbon P. and Bertha E. Dubbs
Fndn.)

720. Measurement of C" of plasma glucose by
a simplified liquid scintillation method.
CuaRA ByJERKNES,* Rospert STEELE AND
Wiii1AM BeErnstTEIn.* Depts. of Biology and
Instrumentation, Brookhaven Natl. Lab., Upton,
N.Y:

As reported previously by Rosenthal and Anger
(Rev. Scient. Instruments 25: 670, 1954) a selected
single photomultiplier tube can be used to count
C™ or H® by the liquid scintillation method.
Using a nonoverload amplifier and scaler com-
bination already described (S1nEx et al., J. Biol.
Chem. 213: 673, 1955), a 14 in. diameter phototube
selected for high signal-to-noise ratio has been
used to count C™ at 67% efficiency in 4:1 xylene-
ethanol containing the phosphor 2-phenyl-5-
(4 biphenylyl) 1,3,4-oxadiazole (PBD). The
background is 90 cpm, of which 30 cpm is photo
tube noise. The phototube in a lead pig is in a
deep-freeze; quartz vials, 1 x 2} in., wrapped in
white filter paper reflectors are placed on the tube
face in a layer of silicone oil by drawing aside a
shutter which shields the phototube from light
when the pig is open. Glass vials exhibit a long-
lived phosphorescence which can be counteracted
by the use of complex electronic equipment in-
volving 2 phototubes operating in coincidence.

222

Use of the single phototube for C' counting
requires quartz vials, which exhibit the phos-
phorescence to a much lesser degree. The simpli-
fied counter described above has been used to
count C™ in glucose isolated as the osazone from
plasma filtrates. About 10 mg of the osatriazole
derivative made from the osazone is weighed
into a counting vial. The osatriazole is kept in
solution in 25 ml of scintillator fluid by the pres-
ence of dissolved H;BO;. (Work supported by the
Atomic Energy Commission.)

721. Enzymatic formation of methylthiol
esters from 3-phosphoglyceric acid. SIMON
Biack AND Nancy G. Wriacurt.* Natl. Insts. of
Health, Bethesda, Md.

Methyl mercaptan, a metabolically active sub-
stance, is formed from methionine in a wide
variety of organisms. Its carbon atom is incor-
porated into a number of compounds in the rat
(Canellakis and Tarver) and it promotes thio-
methyl adenosine formation in yeast cultures
(Schlenk and Tillotson). Recently it was found
that an enzyme from yeast catalyzes the
conversion of CH;SH, 3-phosphoglyceric
acid and ATP to phosphoglyceryl methylthiol
(PO,CH,CHOHCOSCH;), PO, and ADP. Cys-
teine, glutathione, CoA and H.S are inactive as
substitutes for CH;SH in this reaction and
C:H;SH reacts slowly. A_ specific phospha-
tase forms’ glyceryl methylthiol ester
' (CH,OHCHOHCOSCH;) from phosphoglyceryl
methylthiol ester. The unphosphorylated thiol
ester accumulates in crude yeast extracts on
addition of PGA and CH;SH with a catalytic
amount of ATP; it has been isolated and crystal-
lized in 300 mg quantities from such reaction
mixtures (100 ml of extract, 25 mm of PGA, 6 mm
of CH;SH, 5 mm of MgCl, 500 ml vol., 2 hr. in-
cubation, pH 7.0, 30°). In the course of the fore-
going work a 3rd enzyme was found which
catalyzés the phosphorylation of glyceric acid
by ATP to form PGA and ADP. This enzyme is
highly specific for glyceric acid, being unreactive
with glyceraldehyde, glycerol or other hydroxy
acids. Each of the 3 enzymes described, phospho-
glyceryl methylthiol ester synthetase and phos-
phatase and glycerate kinase, has been purified
5- to 10-fold and each has been separated from
the other two. The relation of these enzyme re-
actions to known biochemical processes is still
obscure.

722. Conversion of acetate to Cy steroids by
human adrenal gland slices. Eric Biocu,*
Grecory Pincus AND RaupH I. DorFrMan.
Worcester Fndn. for Exptl. Biology, Shrewsbury,
Mass.

The adrenal cortex elaborates A‘-androstene-
3,17-dione, 118-hydroxy-A‘-androstene-3 , 17-dione

FEDERATION PROCEEDINGS

Volume 1§

and adrenosterone (ROBERTS AND SzEGO, Ann,
Rev. Biochem. 24: 543, 1955). These A‘*-3-ketoneg
may arise from dehydroepiandrosterone, which
heretofore has not been either isolated from or
demonstrated in adrenal tissue. This study is
concerned with adrenal Ci, steroid production,
particularly dehydroepiandrosterone. Slices of
an adrenal gland, obtained at surgery from a
woman with the adrenogenital syndrome, were
placed into a medium containing 10 ml human
serum, 5 mg serum, 5 mg glucose, 0.01 mm sodium
fumarate and 0.5 me sodium acetate-1-C'. To a
2nd preparation of identical composition, 10 1.v,
ACTH were added. Both preparations were in-
cubated for 3 hr. at 37°C with 95% O2-5% CO: as
the gas phase. The incubated samples were ho-
mogenized, exhaustively extracted with organic
solvents and carrier Ci, steroids added. After
solvent removal, the residues were extensively
purified by solvent partition, chromatography
and formation of derivatives. Radiochemical
homogeneity was ascertained by a) crystalliza-
tions; b) paper chromatography; c) 20 transfer
countercurrent partition system and d) formation
of derivatives by chemical and enzymatic means.
A‘ - Androstene - 3,17 - dione, dehydroepi-
androsterone and 118-hydroxy-A‘-androstene-3, 17-
dione, but not adrenosterone, were shown to
have been synthesized. Androstane-3,17-dione
synthesis was also indicated. ACTH increased
Cig steroid production, as evidenced by a 4-fold
increase in total radioactivity of 118-hydroxy
A‘-androstene-3, 17-dione.

723. Mechanism of certain steroid oxidations
effected by adrenal tissues and micro-
organisms. Barry M. Bioom,* Mixa HayaAno,
Akira Sarto,* Davip STONE AND Ratpu I,
DorrMan. Worcester Fndn. for Exptl. Biology,
Shrewsbury, Mass., and Chas. Pfizer and Co.,
Inc. Brooklyn, N. Y.

Recent biodxygenation experiments involving
enzyme systems of both adrenal and microbiologi-
cal origin have afforded a new insight into the
mechanism of steroid hydroxylation processes.
Studies with H.O8 and O,'* have conclusively
demonstrated that molecular oxygen, but not
water, is utilized by adrenal tissue in the C-116-
hydroxylation of steroids (Hayano et al. Arch.
Biochem. and Biophys. In press). Similar results
were obtained with an 118-hydroxylating micro-
organism (Cunninghamella blakesleeana). Coupled
with the earlier finding that adrenal 116-hydrox-
ylase requires TPN or TPNH and oxygen, these
observations suggest a gross mechanistic resem-
blance between certain steroid oxidations and their
biological counterparts involving aromatic and
heterocyclic systems. The observation that en-
zymatic epoxidation occurs with unsaturated
steroidal substrates in known microbiological

a ee ee ee

ume 1§

Ann,
etones
which
‘om or
dy is
iction,
ces of
Tom a
, were
human
sodium
. Toa
10 Lv.
sre in-
CO, as
re ho-
rganic

After
asively
graphy
emical
palliza-
ransfer
mation
means.
droepi-
e-3,17-
wn to
'-dione
reased
-4-fold
ydroxy

ations
micro-
AYANO,
LPH I,
tology,
ud Co.,

rolving
riologi-
to the
cesses.
usively
ut not
C-116-
_ Arch.
results
micro-
‘oupled
:y drox-
, these
resem-
id their
ic and
lat en-
‘urated
logical

March 1956

hydroxylating systems (BLoom aNnp SuHuLL, J.
Am. Chem: Soc. 77: 5767, 1955) leads to the hy-
pothesis that the related hydroxylations proceed
by direct electrophilic attack on the appropriate
carbon-hydrogen bond. Stereochemical arguments
will be offered in support of this proposal, and
the related incubation of unsaturated steroids
(17a,21 - dihydroxy - A+) - pregnadiene - 3,20-
dione) with adrenal tissue will be discussed.

724. Metabolic fate of some _ flavanones.
AtBerT N. Bootu,* Francis T. JonEs* AND
FLroyp DsEps. Western Utilization Research
Branch, Agricultural Research Service, US.
Dept. of Agriculture, Albany, Calif.
Two-dimensional chromatograms of the ether

extracts of rabbit urines before and after oral
administration of hesperetin, homoeriodictyol or
naringenin were sprayed with diazotized sul-
fanilic acid followed by 20% sodium carbonate to
detect phenolic compounds. Administration of
either hesperetin or homoeriodictyol resulted in
excretion of meta-hydroxyphenyl-propionic acid,
which was not present in the control urine. Homo-
eriodictyol also gave rise to dihydroferulic acid.
Administration of naringenin resulted in the
excretion of para-hydroxyphenylpropionic acid,
which was absent from the control urine. In each
case 2 conjugate of the parent substance adminis-
tered was excreted. The meta- and para-hydroxy-
phenylpropionic acids and dihydroferulic acid
were identified chromatographically, the meta
derivative also being identified crystallographi-
cally. It is interesting to note that each of the
3 flavanones suffered molecular cleavage to yield
a phenolic acid with a 3-carbon side chain, whereas
earlier studies (BoorH, Murray, DreEps anp
Jones, Federation Proc. 14: No. 1, March 1955)
showed that the flavenols, rutin and quercetin,
gave rise to phenolic acids with a 2-carbon side
chain. The formation of meta-hydroxyphenyl-
propionic acid from hesperetin must have in-
volved demethoxylation, or demethylation fol-
lowed by dehydroxylation of the number 4’ carbon
before or after splitting of the hesperetin molecule.
Similarly, before or after splitting of the homo-
eriodictyol molecule, there must have been
dehydroxylation of the number 4’ carbon and
demethylation of the methoxy group on the
number 3’ carbon to account for the formation of
meta-hydroxyphenylpropionic acid.

725. Further experiments on sodium and
potassium in single nephrons of Necturus
kidneys. Puytuis A. Borr. Dept. of Physio-
logical Chemistry, Woman’s Med. College of
Pennsylvania, Philadelphia.

Blood serum and tubule fluid collected from
the kidneys of Necturi by the method of Richards
and Walker were analyzed for sodium and po-

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

223

tassium using the dual channel flame photom-
eter (Federation Proc. 14: 185, 1955). Inulin and
chloride were determined in the same samples
when possible. Sodium results are essentially the
same as those obtained previously, using ultra-
micro chemical methods (Trans. 5th Conference
on Renal Function, Josiah Macy Jr., Found.,
1954). Results to date for potassium confirm
reabsorption from the proximal tubule but show
more spread than those reported earlier. Attempts
are being made to detect distal tubule secretion
of potassium under various conditions. For this
serum potassium and inulin values are compared
with those of proximal tubule fluid and ureteral
urine collected simultaneously. (Supported by a
grant from the Life Insurance Med. Research
Fund.)

726. P= and O8 incorporation into intra-
mitochondrial phosphate compounds dur-
ing oxidative phosphorylation. P. D. Boyrr,
L. Ernster* anp O. LinpBerG.* Univ. of
Minnesota, Minneapolis, and Wenner-Gren Inst.,
Stockholm, Sweden.

Studies of the path of inorganic phosphate (P;)
and of phosphate oxygen in the reaction media
do not suffice to establish the primary reactions
of oxidative phosphorylation. By use of a simple
filtration technique, estimations can be made of
the status of intramitochondrial components
during oxidative phosphorylation. In 30-45 sec.
after the introduction of P;**, the specific activity
of the terminal group of intramitochondrial ATP
was over 8 times that of the middle group. In
similar studies with introduction of H.,O™ into
the medium, the O concentration was highest
in the intra-mitochondrial P;, next in the terminal
phosphate of ATP, and lowest in the middle
phosphate of ATP and labile phosphate of ADP.
Related studies show that the rapid exchange of
the phosphate of ADP in the medium with P;
involves principally the terminal phosphate of
the ATP and that the ‘bridge’ oxygen is supplied
by the ADP. Under similar conditions the ex-
change of pyrophosphate-P*? with ATP phosphate
gives equal labeling of both the labile phosphate
groups. These studies give a firmer basis to the
hypothesis that the primary activation of oxida-
tive phosphorylation is principally or entirely
that of the P;, the P,; forming an intermediate
which subsequently donates a phosphate group
to ADP. The results and other considerations
suggest that reversal of the primary activation
reactions will account for the observed rapid
exchange of oxygen between phosphate and H,0.
2,4-Dinitrophenol appears to interfere in the
primary activation of P;.

727. Metabolism of 11-hydroxy-4‘-andros-
tene-3,17-dione-4-C". H. Leon Brap.tow,

224

Leon HeiuMan* anv T. F. GaLLaGuer. From
the Sloan-Kettering Inst. for Cancer Research,
New York City.

Tracer doses of 118-hydroxy-A‘-androstene-3, -
17-dione-4-C'* were administered intravenously
to 2 human subjects. The rate of excretion of the
radioactive metabolites was more rapid and the
recovery of products was more complete than after
hydrocortisone. Thus 80% of the administered
radioactivity was eliminated in the urine during
the first 4 hr. and the recovery was quantitative
after 24 hr. Blood levels of free and conjugated
radioactivity were measured during the initial
periods after injection and the free and conju-
gated steroids in the urine were estimated at
intervals. The major portion of the metabolites
were excreted as glucosiduronic acids. The oxygen
function at C-11 was retained during metabolic
transformation since androsterone and etio-
cholanolone were devoid of radioactivity. The
principal end products isolated were 118-hydroxy
and 11-keto derivatives of saturated 3-hydroxy-
17-ketosteroids of the normal and allo series.
The amounts of each were measured by reverse
isotopic dilution. There were only small amounts
of more polar metabolites.

728. Effect of thiamin deficiency on mamma-
lian erythrocyte metabolism. Myron Brin,
STEPHEN S. SHonet* anp CuHar.es 8S. Davip-
son. Thorndike Memorial Lab., Second and Fourth
(Harvard) Med. Services, Boston City Hosp., and
the Depts. of Biological Chemistry and Medicine,
Harvard Med. School, Boston, Mass.

Studies from this laboratory have demonstrated
that methylene blue activates a hexose mono-
phosphate cycle (HMC) in mammalian erythro-
cytes. The respiratory increment accrues from
glucose oxidation, determined with C'‘-labeled
substrate (Brin and Yonemoto). As transketolase
is essential in this cycle and is thiamin dependent,
its actyvity was used as a test for this vitamin.
One-hundred-twenty rats were made thiamin
deficient by feeding a purified thiamin-low diet
and keeping in wire bottom cages. Erythrocytes,
obtained by cardiac puncture biweekly, were
incubated in the Warburg at 38°C under air with
methylene blue and substrate. Cells from severely
deficient animals (3 wk.) exhibited 30% decreased
respiration, 90% decreased recovery of carbon-2
of glucose and accumulation of pentose to 3 times
the control system. By the time growth ceased
(2 wk.) and before signs of severe deficiency were
evident, the recovery of carbon-2 of glucose as
CO. was decreased 45-55%. Pentose accumula-
tion was reversed often by adding thiamin to
deficient cells in vitro and in 2 instances there was
a concomitant accumulation of a 7-C sugar. All
biochemical aberrations measured were reversed
by in vivo injection of thiamin intraperitoneally

FEDERATION PROCEEDINGS

Volume 1§

or by feeding complete rations. Erythrocytes,
therefore, comprise a tissue in which the HMC
may be studied in the absence of interfering oxi-
dative phenomena. In addition they present
readily accessible biopsy tissue for enzymatic
evaluation of nutritional status.

729. Disposition of Ca“ injected intraperi-
toneally into suckling rats. FELIx BRoNNgER
(introduced by R. M, ArcuiBap). Rockefeller
Inst. for Med. Research, New York City.

One hour after intraperitoneal injection of
calcium chloride-Ca* into 10-day-old rats, the
specific activity of the serum approximated 40%
of the dose per milligram of calcium (%/mg);
by the 108th hr. it had decreased exponentially
to 0.5%/mg. During the same interval of time the
specific activity of the calcium in muscle, in the
pooled internal organs (liver, heart, lungs, gonads,
spleen, kidneys, thyroid gland) and in the pelt
also decreased exponentially, whereas the specific
activity of the skeletal calcium increased up to
the 12th hr. and thereafter decreased slowly. The
specific activities of the calcium in the various
tissues seldom equalled that in serum. For ex-
ample, by the 8th hr. after injection, the specific
activity of the pelt was 1.8 times that of the serum;
by the 85th hr. it had returned to the level of the
serum specific activity. After 12 hr. the specific
activity of the internal organs was about 5 times
that of the serum, and after 36 hr. the specific
activity of the muscle calcium was 1.5 times that
of the serum. These findings are discussed in
relation to the kinetics of calcium metabolism,

730. Inhibition of valine and isoleucine syn-
thesis in yeast by thioctic acid analogues.
Harry P. Broquist AND ARTHUR V. STIFFEY.*
Nutrition and Physiology Section, Research Div.,
American Cyanamid Co., Lederle Labs., Pearl
River, N. Y.

A series of 8-thioloctanoic acids containing
different substituents in the 6-position have been
prepared in these laboratories (J. A. BRocKMAN,
Jr., aND P. Faso. In preparation) and have been
found to inhibit growth of the yeast Torula
cremoris. When 7’. cremoris was grown in a simple
synthetic medium these analogues were markedly
inhibitory for growth but the inhibition was re-
versed competitively by a number of fatty acids
including acetic acid, palmitic acid (as Tween 40),
and oleic acid (as Tween 80). Hence the analogues
may interfere with the utilization rather than the
biosynthesis of fatty acids in this organism. The
toxicity of the analogues for 7’. cremoris was not
reversed by thioctic acid over a wide range of
concentration, but was counteracted by Difco
yeast extract. Fractionation of yeast extract
showed this effect to be due to its content of isd
leucine and valine. Both amino acids must be

=—- = oo

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rocytes,
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tion of
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For ex-
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IFFEY,*
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March 1956

present in substrate amounts to reverse the
analogues for 7’. cremoris, and the reversal was
noncompetitive in nature. The analogues may
interfere with the synthesis of isoleucine and
valine in 7’. cremoris possibly by blocking a com-
mon or similar step on the biosynthetic pathway
of these amino acids.

731. Purification of mammalian tyrosinase.
F, CurisTINE Brown (introduced by A. CLARK
GRIFFIN).

Harding-Passey tumors from BALBC female
mice were subjected to the procedure of Dalton
and Nelson (J. Am. Chem. Soc. 61: 2946, 1939) for
the crystallization of wild mushroom tyrosinase.
The tissue was extracted with water and the
aqueous extract was fractionated with 0.2 and 0.6
saturated (NH,)2 SO, followed by acetone precipi-
tation, adsorption on activated alumina and char-
coal and finally reprecipitation with 0.6 saturated
(NH,)2 SO,. All fractions were dialyzed until free
of SO,~ ion. The tyrosinase activity of the different
fractions was determined manometrically in the
Warburg apparatus using L-tyrosine as substrate.
Activities, expressed in units as proposed by
Hogeboom and Adams (J. Biol. Chem. 145: 273,
1942), in a typical experiment were as follows:
original homogenate, 0.69 u/mg N; 0.2 saturated
insoluble fraction, 2.31 u/mg N; and the final 0.6
saturated insoluble fraction, 5.26 u/mg N. The
latter fraction, possessing the highest activity,
was a slightly turbid light colored solution, indi-
cating that most, if not all, of the melanin had
been removed. Attempts to crystallize the enzyme
from the clean active fraction have not been suc-
cessful to date. However, these results indicate
that mammalian tyrosinase has many of the same
properties of mushroom tyrosinase and may
therefore be subject to purification in the crystal-
line state. The properties and activities of the
mammalian preparation will be compared with
crystalline mushroom tyrosinase.

732. Antigenic structure of ribonuclease.
Ray K. Brown, HELEN Van VUNAKIS AND
LAWRENCE LEVINE (introduced by Mary C.
Panaporn). Div. of Labs. and Research, New
York State Dept. of Health, Albany.

Rabbit antiserum to crystalline beef ribo-
nuclease (RNase) was shown to be immuno-
chemically homogeneous. Ribonuclease a and b,
separated chromatographically, react with the an-
tiserum and either precipitates all of the antibody
from the serum. The diffusion constant for RNase
obtained with the serum is 9.6 X 10-7? cm?/sec.
Specific precipitates contain RNase activity and
when analyzed in the region of antibody excess
indicate that 2 molecules of antibody react with
1 molecule of RNase. The antigenic and enzymatic
activities are resistant to 8 M urea, 20% TCA, or

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

225

boiling for 5 min. at neutral px. This suggests that
the antigenicity of RNase is independent of
secondary bonding. Treatment with performic
acid destroys the antigenicity as judged by
precipitin, complement-fixation and precipitin
inhibition technics, suggesting that the antigenic-
ity depends on intact sulphydryl bridge(s).
Treatment with trypsin, chymotrypsin, pepsin
and hydrolysis with 0.25 M oxalic acid at 100°C
were studied kinetically by assay of enzymic and
antigenic activity. The rate of loss of both activi-
ties is similar.

733. Quantitative studies on the urinary ex-
cretion of anthranilic acid metabolites in
rats and man. R. R. Brown anp J. M. PrRIcE
(introduced by E. C. Miter). Cancer Research
Hosp., Univ. of Wisconsin Med. School, Madison,
Wisc.

The development of quantitative ion exchange
methods for the determination of aromatic amines
in urine (R. R. Brown anp J. M. Price J. Biol.
Chem. In press) made it possible to study the
excretion of such compounds after the administra-
tion of small amounts of anthranilic acid, a metab-
olite of tryptophan. Rats given intraperitoneal
injections of anthranilic acid (0.8-50 mg) excreted
50-75% as free anthranilic acid and 10-25% as o-
aminohippuric acid in the urine. Except at bigh
doses of anthranilic acid, the glucuronide was a
minor metabolite. In rats, 60-99% of the injected
anthranilic acid was accounted for in the first 24
hr. Normal humans given oral doses of 22.5-112.5
mg of anthranilic acid excreted 103-123% of the
dose as 0-aminohippuric acid. Excretion of free
anthranilic acid and its glucuronide was less than
3.6 and 5.0%, respectively. Administration of
anthranilic acid had no effect on the urinary
excretion of kynurenine, kynurenic acid, xan-
thurenic acid or N-methyl-2-pyridone-5-carbox-
amide. Thus, it did not seem to alter the overall
metabolism of tryptophan to niacin. Although
rats and man handle anthranilic acid somewhat
differently, the high percentage of anthranilic
acid accounted for as o-aminohippuric acid or free
anthranilic acid suggests that the anthranilic
acid moiety is not further degraded in these
species.

734. Distribution of polynucleotide phos-
phorylase. D. O. BrummMonp* AnD SEVERO
Ocuoa. Dept. of Biochemistry, New York Univ.
College of Medicine, New York City.

The incorporation of radioactive orthophos-
phate into 5/-nucleoside diphosphates, such as
ADP, IDP or CDP, on incubation of cell and tis-
sue extracts with the above nucleoside diphos-
phates or with the biosynthetic AMP poly-
nucleotide (M. Grunserc-Manaco, P. J.
Ortiz anv 8S. Ocnoa, Science 122: 907, 1955), in

226

the presence of Mg*t, has been used to determine
the occurrence of polynucleotide phosphorylase.
The method is based on the reversible reaction
catalyzed by the enzyme, namely n base-ribose-
P-P = (base-ribose-P), + nP. The enzyme is
widely distributed in bacteria whether aerobic or
anaerobic, gram positive or negative. It has so
far been found in Azot. vinelandii, Alc. faecalis,
Staph. aureus, E. coli, Cl. kluyveri, B. cereus, S.
lactis R, S. haemolyticus, S. faecalis and M. lyso-
deikticus. Negative results were obtained with
L. arabinosus and Cl. acetobutylicum extracts. The
enzyme is also present in yeast (M. GRUNBERG-
ManaGo, personal communication). In higher
plants the enzyme has been detected so far in
ammonium sulfate fractions of spinach leaves.
Low amounts of the enzyme, especially in the
presence of interfering side-reactions, may be
difficult to detect. This may explain the failure so
far to obtain unequivocal evidence for its presence
in animal tissues.

735. Sedimentation and diffusion studies on
hyaluronic acid. RoBert BrunisuH, MARTIN
VANGEROV AND WILLIAM IRVINE (introduced by
JosEpH F. Nyc). Dept. of Physiological Chemis-
try, School of Medicine, Univ. of California, Los
Angeles.

Hyaluronic acid was prepared from bovine
vitreous humor. Sedimentation and diffusion stud-
ies were carried out employing a potassium chlo-
ride, sodium cacodylate buffer at ionic strength
0.3, pH 7.0. A graph of the reciprocal of the sedi-
mentation constant against concentration gave
an extrapolated zero concentration value of 4.0.
The slope of the 1/S20-concentration line was
0.90. It was determined that the sedimentation
constant (after correction to water at 20°) was
temperature dependent. The values for a 0.1%
solution varied from 2.2 at 7°C to 2.9 at 27°C. At
a constant temperature, however, no effect on
the sedynentation constant was produced by
varying the gravitational force. Diffusion analyses
were made employing the Rayleigh optical system
embodied in the Spinco Model H electrophoresis-
diffusion apparatus. The curves obtained were
analyzed by the method of moments and by the
area-height technique in order to obtain both
weight and number average diffusion coefficients.
Certain runs were analyzed by Boltzmann’s
method. The results indicate that hyaluronic acid
is a polydisperse system which shows concentra-
tion dependent diffusion although the concentra-
tion dependence was small compared with that of
other asymmetrical molecules. A temperature
effect was noted here as well as in the sedimenta-
tion determinations. (Supported by The Estelle
Doheny Eye Fndn., Los Angeles.)

736. Preparative chromatography of amino
acids by carrier displacement. DoNa.p L.

FEDERATION PROCEEDINGS

Volume 16

BucHaNaNn. Radioisotope Service, VA Hosp.,

West Haven, Conn.

Heretofore displacement chromatographic
methods have not given quantitative yields be-
cause mixed fractions at zone boundaries were
discarded. Because the order of displacement of
amino acids (a.a.) and other substances on cation
resins is largely determined by relative basicity, a
base with proton affinity intermediate with respect
to 2 a.a. will separate them as they move down a
column. Analogously, acidic substances may func-
tion as ‘carriers’ on anion resin columns. Using
this principle, complete separation of individual
basic and acidic a.a. from each other and from the
neutrals has been achieved. The latter are then
separated into groups. A mixture containing 1
mo each of 15 a.a. is first passed through a cascade
of 3 columns. The Ist, Dowex 1-acetate, retains
the acidic; the 2nd, Dowex 50-pyridine, retains
the basic; and the 3rd, Dowex 50-H, retains the
neutral a.a. Each column is then placed over 1
containing appropriate carriers and developed
with 0.2 m displacing solutions. The a.a. and car-
riers emerge in this order: on the acidic column,
acetic, glutamic, lactic, aspartic and citric; on the
basic column, pyridine, histidine, collidine, lysine,
ammonia, arginine and piperidine. The neutral
a.a. are first developed through Dowex 50-N-iso-
propyl pyrazole. Serine, threonine and _ proline
emerge as a group well separated by the carrier
from alanine, glycine, valine, methionine and the
leucines. These are further subdivided with nico-
tinie acid or pyrazole as carriers. All columns are
8 mm in diameter and from 6 to 50 em in length
and must be heated in some instances. Carriers
are removable by evaporation or solvent extrac-
tion.

737. Evidence for existence of pressor and
oxytocic active peptides in angiotonin
preparations. F. M. Bumpus anp H. J.
ScHwarz (introduced by Irvine H. Page).
From the Research Div. of the Cleveland Clinic
Fndn. and the Frank E. Bunts Educational Inst.,
Cleveland, Ohio.

The comparison of pressor and oxytocic activity
of angiotonin preparations in various stages of
purification led to the conclusion that 2 or more
active peptides must be present. Many workers
have established that crude angiotonin has smooth
muscle stimulating (oxytocic) properties; some
have attempted to assay for the pressor peptide
utilizing this activity. Little attention has been
given to the possibility of there being more than
1 active substance present in all of these prepara-
tions. Parallel assays of pressor and oxytocic
activity of angiotonin preparations were made
during each step of purification. Pressor activity
was assayed on a dog sensitized by section of the
sino-aortic nerves and treatment with tetraethyl-

do
aci
gly

wh
lev

L-a
oxi
wh
col
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739

Jy ~~ hs! et S&S 0

ime 16
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raphic
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etains
etains
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March 1956

ammonium chloride. Oxytocic activity was meas-
ured on a rat’s uterus by the method described by
Schwarz, Masson and Page. The stages of puri-
fication consist of 1) serial fractionation by addi-
tion of sodium chloride; 2) extraction into organic
solvents; and 3) partitioning in a countercurrent
machine between several different solvent systems.
There is evidence for a partial separation of pep-
tides possessing pressor and oxytocic activities
and that these are not artefacts of purification.
oxytocic activity
pressor activity
about 5.0. It can be concluded that at least 2
active factors were present even in highly purified
fractions.

varies from 0.25 to

The ratio of

738. Regeneration of flavin enzymes during
during recovery from riboflavin deficiency.
HELEN B. Burcu Anp A. M. Coss (introduced
by Morris E. Frrepxin). Dept. of Pharma-
cology, Washington Univ. School of Medicine,
St. Louis, Mo.

It has been shown previously that during de-
velopment of riboflavin deficiency there are
marked differences in the rate and extent of fall
in tissue levels of different flavin enzymes and
coenzymes. The rate of regeneration of several
flavin enzymes during realimentation has now
been studied. Riboflavin deficient rats were in-
jected with riboflavin and the riboflavin coenzyme
and enzyme levels were followed for 24 hr. FAD
was synthesized rapidly in liver but more slowly
in kidney. Free riboflavin was very high at 0.5 hr.
but decreased in 2 hr. to the usual low levels found
in tissues. By 24 hr. the riboflavin phosphate was
back to normal in kidney but had only recovered
50% in liver. In liver, glycolate oxidase activity
doubled in 0.5 hr. and quadrupled in 2 hr. p-amino
acid oxidase activity tripled in 0.5 hr. Oxidases for
glycine, xanthine and L-amino acids were re-
generated less rapidly. Liver xanthine oxidase
which had dropped initially to only one-half the
level of weight controls, was, however, restored
by 24 hr. to the weight control level. In kidney,
L-amino acid oxidase, glycine oxidase and xanthine
oxidase showed only small increases in 24 hr.
whereas D-amino acid oxidase attained the weight
control level in 0.5 hr. By 24 hr. the p-amino acid
oxidase was about 30% lower than in the kidney
of the high riboflavin rats. (Supported by the
Nutrition Fndn., Inc., and the Williams-Water-
man Fund for the Combat of Dietary Disease.)

739. Influence of assimilatory quotient, cell
anabolism and growth on photosynthetic
quantum efficiency. DEAN BurRK, GEORGE
Hopspy* aNd JeHU HuntTeR.* Natl. Cancer
Inst., Bethesda, Md., and Mazx-Planck-Inst. for
Cell Physiology, Berlin-Dahlem.

The most fundamental experimental fact of

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

227

photosynthesis is the production of 1 molecule
of oxygen gas by 1 quantum of light absorbed by 1
molecule of cell chlorophyll. The 1:1 stoichiometry
between QO2 and chlorophyll (light-dissociable
oxygen capacity) has been demonstrated by
direct photochemical titration and gas analysis
(cf. Zeit. Naturforsch. 9b: 769, 1954, et seq.) and
steady-state photosynthesis kinetics (Scient.
Monthly 73: 222, 1951). In the experimental 1-
quantum reaction, the assimilatory quotient
(y = CO: absorbed/O:2 produced) usually exceeds
—1 numerically, corresponding to formation of
substances on the average slightly less reduced
than carbohydrate (e.g. phosphoglyceric (—1.20)
or glycollic (—1.33) acid). Such substances act as
substrates in the immediately following light-
induced respiration (back reaction of the photo-
synthetic energy cycle), whose great magnitude
and significance were first conceived of only
after being discovered experimentally (Natur-
wissenschaften 37: 560, 1950). Under optimum
experimental conditions (e.g. catalytic blue light,
pentavalent vanadium, low pu, thin cultures),
the observed quantum requirement for net pro-
duction of O: in the steady-state photosynthetic
cycle at high light intensities need be no more
than 3 hvy/O2 (near thermodynamic perfection),
providing y for the cycle exceeds —1. With further
conversion of the net organic matter produced in
the cycle to progressively more reduced carbo-
hydrate, protein, fat, coenzymes, pigments, etc.,
involved in processes of cell anabolism and growth
secondary to photosynthesis proper, y progres-
sively decreases to values well below minus
unity (e.g. —0.7), and the minimum observable
values of hy/O2 pass from 3 to 4, 5, 6 and more,
as increasing consumption of O» is needed mecha-
nistically or energetically for such incidental
processes (cf. Federation Proc. 12: 620, 1953;
Science 121: 620, 1955). These studies have involved
both thermophilic and conventional Chlorella,
measured manometrically and electrochemically.

740. Role of glutamine in urea production and
protein synthesis in the livers of normal
and 3’ MeDAB-fed rats. Wiiu1AmM T. BuRKE*
AND Leon L. MILER. Atomic Energy Project,
Univ. of Rochester School of Medicine and Den-
tistry, Rochester, N. Y.

The effects of added glutamine on amino acid
metabolism in the isolated, perfused livers from
normal rats and from rats fed a diet containing 3’
MeDAB for 8-12 wk. have been compared with the
effects of added glutamic acid and added am-
monia. 10 we of pi-Lysine-6-C!4 and 500 mg. of
p-glucose were added to the perfusing blood in all
experiments. All supplements were equivalent in
total nitrogen. Addition of glutamine or ammonia
leads to quantitatively similar urea production
in the first 2 hr. of liver perfusion. The 3’ Me-

228

DAB glutamine supplemented livers produce
almost as much urea as do the normal glutamine
supplemented livers. In normal livers only half
as much urea is produced when the perfusing blood
is supplemented with glutamic acid rather than
glutamine. This depression of urea synthesis
when glutamic acid is the nitrogen source is much
greater in the 3’ MeDAB livers. Only one-third
as much urea is produced. The data are compatible
with the idea that glutamine occupies a central
role in urea synthesis and indicate that a bio-
chemical block may occur, before the reactions
involving glutamine, on the pathway of urea
synthesis in 3’ MeDAB livers. Glutamine supple-
mentation is also associated with decreased C!4O,
production and markedly increased C'* incorpora-
tion into liver and plasma proteins in both types
of livers. These data suggest a unique role for
glutamine in enhancing protein synthesis by the
rat liver.

741. Metabolism of fixed nitrogen by Azoto-
bacter vinelandii. D. P. Burma* anp R. H.
Burris. Dept. of Biochemistry, Univ. of Wis-
consin, Madison.

A. vinelandii growing on normal N:2 as its sole
source of nitrogen was supplied with N!5H,*
during its exponential phase of growth. 1, 2 and 5
min. after addition of N'5H,*, samples of culture
were killed with acid. The cells were hydrolyzed
and their constituent amino acids, purines and
pyrimidines were separated on Dowex 50 and
analyzed for N'*. The media were freed from
ammonia, sugar and salts, and then were sub-
jected to unidirectional chromatography to
separate amino acids for N'* analysis. The media
contained few amino acids, but these had a much
higher N'* concentration than in the cells, possibly
because of acid-extraction of the free amino acids
from the cells. Glutamic acid from the medium
had approximately 4 times the N!5 concentration
of any#ther amino acid and 6 times the N!* con-
centration of the cellular glutamic acid. All the
nitrogenous compounds isolated in the 1 min.
experiment had detectable amounts of N!5;
glutamic acid had the highest concentration.
After 1 min., glutamic acid from the cells had
0.096 atom % N!® excess, aspartic acid 0.034,
glycine 0.013, serine 0.029, threonine 0.036, leucine
0.022, arginine 0.004 and the total cell hydrolysate
0.023. This distribution shows clearly that the
formation of glutamic acid from ammonia is a
primary process of nitrogen assimilation in A.
vinelandii. Purines and pyrimidines also are active
metabolites in the azotobacter; the 1 min. cell
hydrolysate gave thymine with 0.002 atom % N'5
excess, uracil 0.028, xanthine 0.023, cytosine 0.031
and adenine 0.011.

742. Wholesomeness of a gamma-irradiated
diet fed torats. C.H. Burns,* L. E. BRowNELL*

FEDERATION PROCEEDINGS

Volume 15

AND H. C. Eckstein. Fission Products Lab,
and Dept. of Biological Chemistry, Univ. of
Michigan, Ann Arbor.

A long-term feeding and breeding experiment
has been conducted to determine the nutritional
value and freedom from toxicity of a diet, fed to
rats, which has received a sterilizing dose of
gamma radiation. Fifty percent of the diet con.
sisted of a canned meat product, and the remainder
of purified casein, corn starch and alpha cellulose,
After receiving 4,000,000 rep of gamma radiation
from a cobalt-60 source, a complete vitamin and
mineral supplement was added and the diet fed
ad libitum. A colony consisting originally of 31
male and 31 female Holtzman strain albino rats
have been raised and maintained for a period
of over 2 yr. on this diet. Equal numbers of con-
trol animals were fed the nonirradiated diet.
Three generations of offspring have been ob-
tained from the parent generation, the animals of
each generation having been bred twice. In addi-
tion to growth and reproduction performance of
the animals of all 4 generations, data on pathology
and blood cell counts for the 1st and 2nd genera-
tion animals, and some biochemical data on 3rd
generation animals will be presented. The evi-
dence at present indicates that aside from some
destruction of vitamins, the radiation-sterilized
diet used is wholesome for rats. (Supported by the
Michigan Memorial Phoenix Project of the Univ.
of Michigan.)

743. Increased incorporation of radioacetate
into adrenal cortisol and corticosterone in
scurvy. S. BuRSTEIN AND E. M. NapbeEt (in-
troduced by C. G. Baker). The Worcester Fndn.
for Exp. Biology, Shrewsbury, Mass. and the
Natl. Cancer Inst., Bethesda, Md.

The increased excretion of cortisol and 6 B-hy-
droxycortisol in scorbutic guinea pigs (BURSTEIN,
DorFMAN AND NabEt: J. Biol. Chem. 213: 597,
1955) led to this study on the incorporation of
radioacetate into adrenal corticosteroids in
scurvy. 0.1 me of Na-1-C'* acetate was injected
(i.p.) into scorbutic (8) and control (6) animals;
15 and 30 min. later their adrenal glands were
removed and extracted with ethyl acetate; ex-
tracts were diluted with nonradioactive cortisol
and corticosterone and chromatographed on
paper in 2 different systems. Specific activities of
cortisol fractions showed little variation after
rechromatography, while those of corticosterone
fractions decreased considerably. Cortisol frac-
tions with high specific activity were tested for
homogeneity by column partition chromatography
on Hyflo-Supercel, crystallizations of the free
steroid, acetylation and rechromatography, and 4
crystallizations from different solvents. During
this purification procedure, no appreciable decline
occurred in the specific activity of the cortisol;

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March 1956

however, with similar procedures, ‘corticosterone’

fractions showed further decline suggesting that
another radioactive product (possibly Reich-
stein’s compound §) was present with corticos-
terone. Scorbutic animals incorporated 3 to
7 times more radioacetate into the cortisol frac-
tions than did their controls (P < .01); smaller
increases were observed in the ‘corticosterone’
fractions. Increased urinary corticoids in late
acute scurvy (NADEL AND SCHNEIDER, Endocri-
nology 51: 5, 1952) may be related to the higher
incorporation of acetate in scurvy; this in turn
may be due to the stress of the vitamin deficiency
and associated with increased ACTH production.

44. Fluoropyruvic acid: toxic and metabolic
effects. Harris Buscu. Dept. of Pharmacology,
Univ. of Illinois College of Medicine, Chicago.
Monofluoropyruvic acid was synthesized by the

method of Blank et al. (J. Chem. Soc., 2190, 1955)

and purified by anion exchange chromatography.

Intravenous or intraperitoneal injection of doses

of 50 mg/kg or greater into rats resulted in death,

preceded by salivation, retching and tonic con-
vulsions. Fluoropyruvic acid is a noncompetitive
inhibitor of lactic dehydrogenase in systems con-
taining DPNH and twice recrystallized rabbit
muscle lactic dehydrogenase (Sigma), although in

a concentration of 0.003 M, fluoropyruvic acid

itself is reduced at a rate 4 that of a equimolar

concentration of pyruvate. At a concentration of

0.003 M each of pyruvate and fluoropyruvate, the

rate of DPNH oxidation was ? that with 0.003 @

pyruvate alone, and increasing concentrations of
pyruvate did not increase this rate. No significant
suppression of lactic dehydrogenase activity was
produced by alpha- and beta-chloro- and bromo-
propionic acids, acrylic, butyric, bromopyruvic,
diglycollic, fluoroacetic, beta-iodopropionic, ita-
conic, lactic, malic, nicotinic, oxaloacetic and
thiomalic acids, potassium fluoride, acetone,
acetamide and acetophenone. Oxalic acid, a very
potent inhibitor of lactic dehydrogenase, (N1E-

LANDS, J. Biol. Chem. 208: 225, 1954) was ap-

proximately equal in effect to fluoropyruvic acid.

In tissues of animals treated with fluoropyruvic

acid (150 mg/kg) no significant increase in pyru-

vate or lactate was found. However, the concen-
traction of citrate increased in brain, heart,

kidney and spleen from 0.18, 0.10, 0.12 and 0.25

um/gm, respectively to 1.0, 0.85, 3.2 and 2.9 um/gm,

respectively. These results indicate that fluoro-
pyruvate is converted to fluorocitrate which
blocks the citric acid cycle (PETERs et al., Nature

171: 1111, 1953). (Aided by grants from the Public

Health Service and the Jane Coffin Childs Fund.)

45. Biosynthesis of uracil nucleotides. E. 8.
CANELLAKIS (introduced by H. Tarver). Dept.
of Pharmacology, Yale Univ., New Haven, Conn.
Uracil is incorporated into the RNA of normal

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

229

rat liver slices when the concentration of this
pyrimidine in the medium is increased beyond
the levels required to saturate the enzyme systems
which degrade free uracil (CANELLAKIS, Federa-
tion Proc. 14: 324, 1955). The present study shows
that enzyme systems exist in livers of normal
rats which can convert uracil to the nucleotide
stage. The first enzyme involved in this conversion,
uridine phosphorylase, has been partially purified
and shown to catalyze the reversible reaction
uracil + ribose-l-phosphate = uridine + phos-
phate. Cytidine is inactive in this system. En-
zymatic conversion of uridine to the nucleotides
UMP, UDP, UTP and an unidentified uridine
containing compound has been shown to occur
in the presence of ATP. Thus enzymatic path-
ways for the biosynthesis of uracil nucleotides
from the free base exist in normal rat liver; how-
ever, at the present time the extent of their
participation in normal metabolism is conjectural.
A nucleotide pyrophosphorylase reaction, which
would permit the direct synthesis of UMP from
uracil and phosphoribosylpyrophosphate (PRPP),
has not been found in rat liver; however this reac-
tion has been demonstrated in extracts of 2 strains
of Lactobacillus bulgaricus.

746. Methionine activating enzyme. G. L.
CaNToNI AND J. Duretu.* Lab. of Cellular
Pharmacology, Natl. Inst. of Mental Health,
Bethesda, Md.

It was reported earlier that in the reaction be-
tween L-methionine and ATP catalyzed by the
methionine activating enzyme (MABE), all 3 phos-
phates of ATP are mineralized. In the partially
purified enzyme used earlier, the ratio of MAE to
pyrophosphatase was about 0.1. With further
purification the ratio increased to between 5 and
10. With this preparation of MAE, it was found
that: a) the synthesis of S-adenosylmethionine
(AMe) is stimulated by addition of crystalline
yeast pyrophosphatase; b) inorganic pyrophos-
phate inhibits at low concentrations (0.001m or
less), and c) inorganic pyrophosphate accumulates
during the reaction in nearly equimolar amounts
with AMe and orthophosphate. The origin of the
pyrophosphate was investigated by means of
ATP labeled with P*, That ratio of the specific
activity of the pyrophosphate formed during the
reaction to the specific activity of the 6 and y
phosphates in ATP was found to be close to 0.5
when ARP-P*2-P2 was used. Furthermore when
ARP®-P-P was used, more than 90% of the isotope
was recovered in the pyrophosphate formed.
These results clearly indicate that the activation
reaction may be formulated as follows: L-methio-
nine + A-R-P*-P*-P — AMe + P*-P* + Pj.

747. Action of arginase on peptides containing
arginine. FrepERICK H. CARPENTER AND

230

WELLINGTON C. PrerRce.* Dept. of Biochemistry,

Univ. of California, Berkeley.

In an attempt to determine whether or not
arginase could be used to detect the presence of
C-terminal arginine in a peptide chain, the follow-
ing compounds, which were synthesized using the
p-nitrobenzyloxycarbonyl (PNBC) radical as a
blocking group (GisH AND CARPENTER, J. Am.
Chem. Soc., 75: 5872, 1953), were subjected to the
action of beef-liver arginase preparations (15
u/mg): u-leucyl-L-arginine acetate,* L-arginyl-.-
leucine acetate,* L-arginyl-L-glutamiec acid,*
L-argininamide dihydrochloride,* PN BC-t-leucy]-
L-arginine, N*-PNBC-.-arginyl-.-glutamic acid
and Ne*-PNBC-t-arginine. The action of the
enzyme preparation was followed not only by
measuring the rate of release of urea (determined
with urease) but also by determining the products
by paper chromatography. Urea was released, at
least in part, from those compounds marked with
an asterisk. However, chromatographic analyses
showed that with the production of urea there was
a previous or concomitant hydrolysis of the
peptide bonds in these substrates. Rate studies
on L-arginyl-L-glutamic acid indicated that the
peptide bond was split previous to the release of
urea. The arginase preparation did not release
urea or split the peptide bonds in the unstarred
compounds. However, glycyl-i-leucine was hy-
drolyzed to glycine and leucine by the arginase
preparation. The results indicated that arginase
did not act on arginine contained in peptides
until free arginine was liberated by a peptidase
present in the enzyme preparation. (Supported in
part by a grant from Eli Lilly and Co. and grant
A-608C of the Natl. Inst. of Arthritis and Meta-
bolic Diseases.)

748. Synthesis of 6-succinylaminopurine and
structure of adenylosuccinic acid. C. E.
Carter. Dept. of Pharmacology, Yale Univ., New
Haveg, Conn.

The aglycone of adenylosuccinic acid, 6-suc-
cinylaminopurine, has been synthesized from
6-chloropurine and aspartic acid. The reaction
was conducted in aqueous solution at pH 9.5 and
in the presence of 0.2 m excess aspartic acid. The
product was precipitated from solution with a
large volume of ethanol and purified by charcoal
chromatography. Synthetic 6-succinylaminopurine
and the aglycone derived from adenylosuccinic
acid by mild acid hydrolysis are chromatograph-
ically identical in several solvent systems and on
ion exchange resins. Titration data for the natural
and synthetic compounds are the same and the
influence of pH on ultraviolet absorption spectra
for the 2 compounds are in exact correspondence.
The reaction of 6-chloropurine with amino acids
has been extended to cysteic acid, glutamic acid,

FEDERATION PROCEEDINGS

Volume i§

glycine and lysine. All of the 6 substituted puriny|
amino acids exhibit an absorption maximum at
275 my in acid and the aspartic, glutamic and
cysteic acid compounds are readily degraded in
acid to a diazotizable amine. This lability of the
substituted purines to acid degradation is be-
lieved to involve an intermediate compound
arising from the cyclization of the carboxyl sub-
stituent to an imidazole nitrogen. Adenylosuccinig
acid and its aglycone exhibit similar lability to
acid and the resultant diazotizable amine ig
chromatographically identical with that derived
from acid degradation of 6-succinylaminopurine,
These studies indicate that the aglycone of
adenylosuccinic acid is 6-succinylaminopurine
and support the structure originally proposed for
the compound, (6-(succinylamino)-9-(ribofura-
nosy! 5’-phosphate)-purine).

749. Water balance in the rat following total
body x-irradiation. W. O. CasTER* AnD
W. D. Armstrone. Dept. of Physiological
Chemistry, Univ. of Minnesota, Minneapolis.
The water balance of groups of 350 gm male

white rats was studied before and after irradiation
with an Lbs dose (700r) of x-ray. The fluid
changes studied include: a) total body water
balances obtained by metabolism methods and
body analysis; and b) estimates of the total volume
and tissue distribution of the plasma, extracellular
and intracellular fluid spaces as determined by
dilution and tissue analysis methods. Following
irradiation, the loss of water roughly parallels
the loss of body weight. During the first 2-4 days
of the period there is a relative increase in total
body thiocyanate space which, however, is not
paralleled by an increase in the relative chloride
space in any of the major tissues. This can be
explained by the facts that: 1) most of the meas-
ured thiocyanate space is in skin, supportive
tissue and the water of the gastrointestinal con-
tent, while the intracellular space is largely in the
muscle mass; and 2) following irradiation there isa
rapid loss of muscle mass, but an increase in
gastrointestinal water. These data provide an
opportunity for the direct comparison of sodium,
chloride and thiocyanate spaces in the different
tissues. During the first day post-x-ray, the
chloride space decreases without a corresponding
change in sodium space. At 6 days there is a large
parallel increase in the chloride and Evans blue
spaces in several tissues.

750. Effects of salt on structure of deoxy-
pentose nucleic acid. LIEBE F. CAVALIERI,
Morton Rosorr* aND BARBARA ROSENBERG.*
Sloan-Kettering Inst. for Cancer Research, New
York City.

Recently, Thomas (Biochim. et Biophys. Acta,

tr:
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num at
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z total
* AND
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Js and
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LIERI,
3ERG.*
, New

Acta,

March 1956

14: 231, 1954) showed that an irreversible change
(‘denaturation’) takes place when DNA is exposed
to very low salt concentrations or when it is
dissolved in water. We have investigated the
effects of this change on optical density, vis-
cosity, streaming dichroism and light-scattering.
It appears that the minimum salt concentration
which will prevent irreversible change (at pH
6.5) is ~ 10°? m. DNA which has never been
exposed to lower salt concentration obeys Beer’s
Law at all DNA concentrations while DNA dis-
solved in water does not do so in the same con-
centration range. A parallel variation in intrinsic
viscosities has been observed. The intrinsic
viscosity (measured in 0.2 m salt) of undenatured
DNA is about 5 times greater than that of the
denatured. The dichroic ratio of completely
denatured DNA is 1.0, while that of undenatured
material is 1.5, which shows that the chromo-
phores are no longer ordered and that the struc-
ture is collapsed. This is supported by light-
scattering results which indicate a contraction
(~40%) of the denatured molecule, but no change
in molecular weight. DNA may also be dissolved,
without damage, in water at pH 7.5 but denatura-
tion occurs when the pH is then lowered to 6.5. A
relation between this type of denaturation and
biological activity has been shown by the loss of
transforming activity of pneumococcal DNA
when exposed to salt lower than 10-* m; however,
its activity was essentially constant at salt con-
centrations above 107? m.

751. Cytochrome b, and b; systems. BRITTON
CuHaNcE, MARTIN KLINGENBERG* AND ENZO
Borrr.* Johnson Fndn., Univ. of Pennsylvania,
Philadelphia and Univ. of Ferrara.

Cytochrome 6b; of microsomes and cytochrome
bs, the purified ‘flavohemoprotein’ (R. K. Morton,
Nature 173: 799), are members of the group of ‘b’
hemoproteins that are believed to mediate the
2 to 1 electron transfer from flavoprotein to cyto-
chrome c (or to c:). The hemoprotein parts of
bs and be are found to have very similar spectra,
reactivities towards ferricyanide, autoxidiza-
bility, ete. But they differ from b of the respiratory
chain, especially in their spectra at liquid air
temperature: 6;’’ and b2’’ show a split a band
with peaks close to 553.4 and 558 my while b’’
shows a single peak at 559 my. The spectra of the
b, and b; systems differ at 460 my presumably
because of their different flavoprotein parts.
Both hemoproteins can be titrated in a one-
electron-per-iron-atom reaction by using DPNH
for b; and L(+)lactate for be, the respective slopes
of the titration being Ae = 16 or 21 em™mm™!
for their a bands. Back titration with ferricyanide
gives lower values of Ae (about 4 for b;) suggest-
ing that electrons can be donated from com-

we
eas 242

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

231

ponents of the system other than hemoprotein.
This reaction may explain 2 to 1 electron transfer
and electron transport in nonphosphorylating
respiratory chains in which cytochrome b does not
react rapidly enough to participate. On the other
hand, in the presence of excess reducing substrate,
additions of relatively high concentrations of
ferricyanide give cycles of oxidation and reduc-
tion of the hemoprotein parts of bz and 6; so closely
synchronized with the kinetics of ferricyanide
reduction that the hemoprotein may be tenta-
tively regarded as ‘Michaelis Compound’ in the
overall activity.

752. Factors affecting diphosphopyridine
nucleotide concentrations of liver. CAROL
C. CHane,* Howarp M. Rawns.ey,* F. V.
FLYNN* AND JoHN G. RetnHOLD. Pepper Lab.,
Univ. of Pennsylvania, Philadelphia.
Diphosphopyridine nucleotide concentrations

have been measured in livers of mice in order to

evaluate the importance of age, sex, fasting and
liver necrosis caused by carbon tetrachloride

administration. Homogenates were prepared im-

mediately after excision of the liver and DPN

splitting enzymes inactivated by heating. Oxi-
dized (DPN) and reduced (DPNH) diphospho-
pyridine nucleotides were measured with the aid

of yeast aleohol dehydrogenase at pu 8.8 and 7.0

by change in absorbancy at 340 mu in the presence

of ethanol or acetaldehyde. Experiments were
designed so that each observation was controlled
by simultaneous study of a litter mate. Differ-
ences between means were evaluated by means
of the ¢ test. In female mice of the National

Institute of Health Strain, concentrations of

DPNH and the sum of DPN and DPNH were

significantly higher than in males (P less than

0.02 and 0.05, respectively). A trend toward

higher DPN and DPNH concentrations in 3-

month-old as compared with 1-month-old mice

was noted. Mice deprived of food for 48 hr. had
lower DPN (P less than 0.01) and higher DPNH
concentrations than mice having access to food,
although the difference in DPNH was not sig-
nificant at the 5% level. Markedly decreased
concentrations of DPN and DPNH were found

48 hr. after carbon tetrachloride was given by

stomach tube. (Supported by the Office of the

Surgeon General, U. S. Army.)

753. Cortisone inhibition of chondroitin
sulfate synthesis in guinea pigs fed large
quantities of ascorbic acid. CHUAN-HUAN
Wvu CuHenc* anp Ropert E. SHanx. Nutrition
Lab., Dept. of Preventive Medicine, Washington
Univ., St. Louis, Mo.

A striking inhibitory effect of cortisone acetate
on chondroitin sulfate synthesis in costal cartilage

232

has been observed in gulnea pigs on relatively
high levels of intake of ascorbic acid. Three groups
of young guinea pigs were maintained on a scor-
butigenic diet and given daily supplements of 0.0,
0.7 and 50.0 mg ascorbic acid per 100 gm body
weight, respectively. Half of the animals in each
group received 2 mg cortisone acetate per 100
gm/day for 4 days before killing on the 16th day
of the diet. Chondroitin sulfate synthesis was
measured by the incorporation of S*5 into costal
cartilage 24 hr. after a dose of carrier-free NazS*5O,.
The data indicated that guinea pigs fed ascorbic
acid at the 0.7 mg level (the minimal daily re-
quirement) synthesized twice as much chondroitin
sulfate in costal cartilage as those receiving no
supplement. Further increase in chondroitin
sulfate synthesis when the 50.0 mg level of intake
of ascorbic acid was given did not prove to be
significant. Cortisone acetate was without effect
on chondroitin sulfate synthesis with low levels of
ascorbic acid intake, but depressed the rate of S*5
incorporation into cartilage of animals fed 50.0
mg/100 gm/day to values comparable to those
observed in scorbutic animals. The possibility
that this suppressive effect resides in the syner-
gistic action of ascorbic acid and cortisone on
certain enzyme systems as well as other mecha-
nisms of action are to be considered.

754. Dinitrophenol and the site of respiratory
lesion in dietary necrotic liver degenera-
tion. Srmpney S. CHERNICK AND K1LaAus
Scuwarz.* Natl. Inst. of Health, Bethesda, Md.
A respiratory lesion in liver slices from rats fed

a vitamin E-free Torula yeast diet has been

described (S. CHernick, et al., J. Biol. Chem.

217: 829, 1955). The rate of oxygen consumption

markedly declines after an initial period of normal

respiration. These investigations suggested that
the defect did not reside directly in the chain of
carbon, metabolism but rather in an ancillary
system such as oxidative phosphorylation. The
effects of 2,4-dinitrophenol (DNP) in vitro on
the respiration of such liver slices were investi-
gated. O2 consumption of normal livers is either
increased or decreased by DNP, depending on
its concentration and other factors, e.g. glycogen
content of the slices (H. NEIMEYER AND E. Fia-

vEROA, Arch. Biochem. and Biophys. 54: 135,

1955). When slices of deficient livers were exposed

to concentrations of DNP ranging from 10-* to

50 X 10-5 M, regardless of the initial glycogen

content of the slices, the stimulatory and in-

hibitory effects of the drug were masked by the
failure in O2 uptake. This infers that the site of
the respiratory lesion is separate and distinct from
that of DNP action; it may involve a defect in
ATP synthesis.

FEDERATION PROCEEDINGS

Volume i§

755. Virtual renal functional volumes. Fray.
cis P. Cu1InaRD AND THEODORE Enns. Depis,
of Medicine and Physiological Chemistry, Johng
Hopkins Med. School and Med. Div., Baltimore
City Hosps., Baltimore, Md.

The technique for determining renal excretion
patterns in dogs (Am. J. Physiol., 180: 617, 620;
182: 247) provides data (mean transit times and
urine flows) which permit calculation of virtual
intrarenal volumes of distribution available to
various substances with essentially uni-modal
patterns. The volume for glomerular substances,
Va, varies directly with urine flow, F. The volumes
for sodium and chloride ions, Vna and Voq, are
nearly equal, substantially less than Va, and show
similar variations with F. The difference, Vg -
Vna, Shows smaller variation with F and generally
is between 0.5 and 2.0 ml. The volume for secreted
PAH, Vpans, is the largest found. The difference,
Veans — Va, is larger than Vg — Vna, increases
little with increasing F, and varies between 15
and 3.0 ml. Values for urea, Vurea, are intermediate
to Vg and Vpans. The difference, Vurea — Vo,
varies between 0.5 and 2.0 ml. If the hypothesis of
back diffusion of urea in the distal segments is
correct, the difference, Vurea — Va, may providea
measure of the volume of the distal segment cells,
Vpans — Va may provide a measure of the volume
of cells involved in PAH secretion and Vg — Vy
may provide a measure of the volume of the
tubular and glomerular lumen proximal to the
zone of addition of sodium to the urine.

756. Effect of excess dietary methionine on
iron metabolism in the rat. Haro. C.
Cuortz* AND CLARENCE P. Bere. Dept. of Bio-
chemistry, State Univ. of Iowa, Iowa City.

The excessive addition of pi-methionine to a
diet which promotes growth in the albino rat is
known to cause growth-retardation and excessive
deposition of hemosiderin in the spleen (VAN
PiusuM AND Bera, J. Biol. Chem. 183: 279, 1950).
Erythrocyte counts and hemoglobin concentra-
tions have subsequently indicated a slight anemia
in rats fed a diet supplemented with 2.0% pt-
methionine, as compared with rats fed the same
diet supplemented with only 0.2% pi-methionine.
The anemia was abolished by the further addition
of 0.048% CoSO,-7H:0 to the diet, as evidenced
by an increase in the red cell counts and hemo-
globin concentrations to normal levels. However,
the addition of the cobalt did not arrest the
growth-retardation. Addition of the same amount
of cobalt to the diets of the rats receiving the
0.2% pu-methionine supplements produced a
polycythemia. High concentrations of total
spleen iron were found in the animals receiving
the 2.0% pi-methionine supplement, less in those
fed this supplement plus cobalt, still less in those

inl
Fe
Chi
801
chi

hoi
ter
ant
an

plume If

. Fray.
. Depts,
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«cretion
17, 620;
nes and
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able to
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rolumes
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erence,
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een 15
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ne on
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e toa
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(VAN
1950).
entra-
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st the
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‘ed a
total
eiving
those
those

March 1956

receiving the diet with only the 0.2% pu-methio-
nine supplement. The excessive deposition of
hemosiderin in the spleens of rats fed the 2.0%
pL-methionine supplement apparently results from
an increased breakdown of the erythrocytes.
Surgical removal of the spleens from such animals
did not alter the blood picture or the inorganic
iron content of the heart, kidney, skeletal muscle,
or small intestine. However, it did increase the
inorganic iron content of the liver.

151. Effects of sterols on water and electrolyte
excretion. Date A. CLARK (introduced by
Attron C. Kurtz). VA Hosp., McKinney,
Texas, and Dept. of Biochemistry, Univ. of Texas
Southwestern Med. School, Dallas.

Cholesterol can be utilized by the adrenal
cortex in the biosynthesis of adrenal steroid hor-
mones (HECHTER AND Pincus. Physiol. Rev. 34:
459, 1954). Several sterols have been reported to
inhibit cholesterol utilization in insects (NOLAND.
Federation Proc. 12: 251, 1953) or in the absorption
of cholesterol from the gut (SIPERSTEIN et al.
Circulation 7: 37, 1953). This paper is to report
some studies made to determine whether certain
cholesterol derivatives may act as antimetabolites
of cholesterol to inhibit production of steroid
hormones normally biosynthesized from choles-
terol. Thiocholesterol (38-mercapto-5-cholestene)
and 3-mercapto-5-pregnene-20-one were prepared,
and their effect on adrenal function was evaluated
by a modified water intoxication test. Two groups
of rats were fasted 24 hr. and given intraperitoneal
injections of sterols. A quantity of water equal
to 6% of the rat’s body weight was administered
every hour for 5 hr. Urine volume was recorded at
intervals and the sodium and potassium content
was determined. In the first experiment, rats in
group A received injections of thiocholesterol,
and rats in group B received cholesterol. In the
second experiment, group A rats received cho-
lesterol and group B rats received thiocholesterol.
It was found that rats receiving thiocholesterol
excreted significantly less potassium and more
sodium than rats receiving cholesterol. (Supported
in part by a grant from the Natl. Cancer Inst.,
Natl. Insts. of Health, PHS).

758. Influence of low concentrations of potas-
sium ion upon carbohydrate metabolism in
isolated rat diaphragm. Donatp W. CLARKE
(introduced by A. M. FisHer). Dept. of Phys-
tology, Univ. of Toronto, Toronto, Canada.
Previous workers have shown that a muscle

(rat diaphragm) incubated in a medium which

contains glucose will take up less glucose, and will

show a smaller net glycogen synthesis as the con-
centration of potassium ion in the medium is
raised. With liver slices, on the other hand, an
increase in potassium ion concentration increases

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

233

the amount of glycogen which is synthesized.
In the experiments reported here, glucose uptake
and glycogen synthesis in the rat diaphragm
were measured in media in which various low
concentrations of potassium ion were used. The
effect of added insulin was also measured under
these conditions. It was found that glucose uptake
was first increased, and then subsequently de-
creased, as the potassium ion concentration in the
medium was raised. The maximum effect was in
the neighborhood of 0.01 M, potassium ion. There
was no significant change in the ‘insulin effect’ as
the media were altered. Glycogen synthesis was
not significantly increased, though a suggestive
trend was noted. It is suggested that there may be
at least 2 steps which are affected by potassium
ion in the change from extracellular glucose to
intracellular glucose-6-phosphate. Potassium may
inhibit one of these steps and may stimulate the
other. The different effects seen in various tissues
may not be a result of greatly different metabolic
reactions, but may depend on which of the 2
steps in the particular tissue which is being
studied contains the rate-controlling reaction.

759. Effect of certain benzimidazoles and some
structurally related compounds upon azo
dye destruction by liver homogenates.
C. C. Cuayton (introduced by J. C. Forsss).
Dept. of Biochemistry, Med. College of Virginia,
Richmond.
2,5-Dimethylbenzimidazole (J) and 2-ethyl-5-

methylbenzimidazole (J7) when fed to rats along

with the azo dye, 3’-methy]l-4-dimethylaminoazo-
benzene, prevented liver tumor formation (Cuay-

TON AND AspBortT. Federation Proc. 14, 194, 1955).

In order to obtain some information on the pos-

sible mechanism of this protection, the effect of I

and JI upon azo dye destruction by liver homo-

genates was investigated. Using 0.5 ml of a 10%

rat liver homogenate and 60 ug of the carcinogenic

azo dye 4-dimethylaminoazobenzene, it was found
that the addition of 1 mg of J or IJ decreased ap-

preciably the amount of dye destroyed during 30

min. incubation at 37°C. The amount of J or JI

could be decreased to 250 ug./flask with consistent
inhibition while with 125 yg/flask results were
variable. 5,6-Dimethylbenzimidazole at a level of

1 mg also inhibited dye destruction, but not to as

great an extent as the others. Unsubstituted

benzimidazole, 2-methylbenzimidazole, 5-methyl-
benzimidazole, and 2-mercaptobenzimidazole had
little or no effect. The much greater effectiveness
of the 2,5-dimethyl and the 2-ethyl-5-methyl
derivatives of benzimidazole on inhibition of azo
dye destruction is similar to results noted pre-
viously for these compounds on inhibition of the
incorporation of N15-glycine into heme (ABBOTT
AND Dopson. Proc. Soc. Exper. Biol. & Med. 86:

234

475, 1954; J. Biol. Chem. 211: 845, 1954). A number

of structurally related compounds, including
certain benzotriazoles, benzoxazoles, benzo-

thiazoles and indoles, were tested as inhibitors
in the azo dye-destroying system. Of these,
3-methylindole, 2-chlorobenzothiazole and 2-
phenylbenzothiazole were found to effectively
inhibit azo dye destruction. (Supported in part
by grant, C-1541, from the Natl. Insts. of Health,
PHS.)

760. Effect of gonadectomy on growth and
cholesterol metabolism in the rat. RIicHARD
D. CoLEMAN,* Yu MIn CuHEn,* Rostyn B. ALFIN-
SLATER AND Harry J. DEUEL, JR. Dept. of
Biochemistry and Nutrition, School of Medicine,
Univ. of Southern California, Los Angeles.

The effect of gonadectomy has been studied
in male and female rats as reflected by growth
and changes in cholesterol concentration in the
plasma, liver and adrenal gland when the animals
were placed on varying experimental diets. When
rats which were gonadectomized at weaning were
placed, at maturity, on diets containing 15%
cottonseed oil or 15% cottonseed oil plus 1%
cholesterol for 6 wk., there were no significant
differences in cholesterol concentration in the
tissues studied when compared with intact con-
trols on identical diets. However, when male
rats which were gonadectomized at weaning were
placed on a fat-free diet for 20 wk. to insure
essential fatty acid deficiency, the cholesterol
concentration in the liver was much decreased
over the elevated concentration normally found
in the livers of intact male rats on essential fatty
acid deficient diets. In the female rat, the situa-
tion was reversed; the liver cholesterol concentra-
tion of the gonadectomized female was sig-
nificantly higher than the normal value which
obtained when intact female rats were fed the
EFA deficient diet. In the case of male rats
gonadeftomized after sexual maturity, and placed
on a diet containing 15% cottonseed oil and 1%
cholesterol for 6 wk., there was a marked decrease
in the cholesterol concentration of the liver as
compared with values obtained on the livers of
nonoperated animals on the same diet; in
gonadectomized adult females, the cholesterol
concentration in the liver increased over the
unoperated female rats. Irrespective of the diets
fed, the effect of gonadectomy was to decrease the
rate of weight gain in male rats and to increase
the rate of weight gain in female rats when com-
pared with intact animals. (Supported by a grant
from the Natl. Insts. of Health, PHS.)

761. Purification of thyrotrophic hormone.
P. G. Conpurrre* anp R. W. Bartss. Nail.
Insts. of Health, PHS, Bethesda, Md.

FEDERATION PROCEEDINGS

Volume if

Thyrotrophic hormone (TSH) was extracted
from beef anterior pituitaries with 2% NaCl soly.
tion at pH 7 at 5°C. The saline extract was frae.
tionated with acetone at pH 4. Most of the TSH
was soluble in 50% acetone and was precipitated
by 75% acetone. This precipitate was 40 time
as potent as the starting material and accounted
for 80% of the original activity. This fraction
obtained by precipitation with 75% acetone was
placed on a carboxymethyl cellulose (CM-W)
column (PETERSON AND SoBER. J. Am. Chem. Soe,
76: 1711, 1954) at px 6.2 in 0.01 Mm sodium phosphate
buffer. The TSH was retained by the column,
while the bulk of the contaminating protein wag
not adsorbed and passed through with the first
hold-up volume of solvent. The TSH was then
recovered quantitatively by applying a gradient
of increasing ionic strength, or a combination of
increasing ionic strength and pu. The portion of
the eluate, which contained the TSH, was de-
salted on a mixed bed resin column (Amberlite
MB-3) and was then lyophilized to yield a white
powder. The final product was approximately 20
times as potent as the starting material. Experi-
ments with Amberlite IRC-50 failed to give more
than 50% recovery of TSH, in contrast to the
quantitative recovery from CM-W, although the
elution patterns were qualitatively — similar,
Thyrotrophic activity was assayed in chicks by the
depletion of I'*! in the thyroid (BATES AND Corv-
FIELD. J. Clin. Endocrin. & Metabolism 15: 838,
1955). Preliminary amino acid analysis of the
most active fractions indicated that there was no
cystine or methionine present.

762. Distribution and biosynthesis of indi-
vidual fatty acids in tissues of intact rats
given C!‘-acetate. JoHN G. ConiGuio (intro-
duced by C. 8. Rosinson). Dept. of Biochem-
istry, Vanderbilt Univ. School of Medicine, Nash-
ville, Tenn.

The distribution and biosynthesis of fatty acids
in the rat under various experimental conditions
are being studied. Fatty acids were isolated
from tissues of adult male Sprague-Dawley
rats killed 5 min. after intraperitoneal injection of
CH;C'4OONa. The saturated fatty acid fraction
was distributed in a 100-tube countercurrent dis-
tribution apparatus and recycled to a total of
600-700 transfers. Iso-octane was used as the upper
phase and a mixture of equal volumes of glacial
acetic acid and acetonitrile as the lower phase.
Only palmitic and stearic acids were found in
liver and intestine. In liver these were present
in about equal amounts; in the intestine there was
twice as much palmitic as stearic acid. These
relative proportions were not altered in either
organ after 72 hr. of starvation. The ratio of the
total radioactivity of palmitic acid to stearic in

fra

olume if

xtracted
.Cl solu.
vas frac.
he TSH
‘ipitated
10. times
‘counted
fraction
one wag
(CM-W)
em. Soe,
osphate
column,
fein wag
the first
as then
zradient
ation of
rtion of
was de-
nberlite
a white
tely 200
Experi-
ve more

to the
ugh the
similar,
s by the
» Corn-
15: 838,
of the
was no

. indi-
ct rats
(intro-
tochem-
, Nash-

y acids
ditions
solated
Dawley
‘tion of
raction
nt dis-
otal of
> upper
glacial
phase.
und in
yresent
sre was
These
either
of the
aric in

March 1956

the liver wag 4:1 in the fed group and 1.4:1 in the
starved group. In the intestine the palmitic:
stearic total radioactivity ratio was 2.5:1 in the
fed and 1:1 in the starved group. The change in
ratios in both organs after starvation indicates a
relative increase in the synthesis of stearic com-
pared to palmitic acid. Only small amounts of
stearic acid were found in the saturated fatty acid
fraction of both groups of rats. The predominant
saturated acid present was palmitic. The amount
of radioactivity contributed by the stearic acid
fraction was not significant quantitatively. (Sup-
ported in part by Atomic Energy Commission
Contract no. AT (40-1)-1033.)

763. Reactions of keto acids catalyzed by
plant enzyme preparations. Hric E. Conn
anp 8. Louise Sexr.* Dept. of Plant Biochem-
istry, Univ. of California, Berkeley.

A survey has shown that enzyme preparations
obtained from higher plants catalyze several reac-
tions involving a-keto acids and reduced diphos-
phopyridine nucleotide (DPNH). Thus, phosphate
buffer extracts prepared from the cotyledons
of etiolated lupine seedlings and from potato
tubers contain lactic dehydrogenases active with
DPNH and pyruvie acid. Mitochondria obtained
from lupine cotyledons catalyze the rapid enzy-
matic oxidation of DPNH in the presence of
phenylpyruvic acid (1.3 X 107? m). Since phenyl-
pyruvic acid is known to function as a substrate
for lactic dehydrogenase it was necessary to
determine whether a lactic dehydrogenase as-
sociated with mitochondria is responsible for
the reaction observed. In addition, glyoxylie acid
reductase occurs widely in higher plants and cata-
lyzes the reduction of the two a-keto acids, gly-
oxylic and hydroxypyruvic. However, these
enzymes do not appear to be responsible for the
reaction between phenylpyruvate and DPNH
since neither pyruvic acid nor glyoxylic acid is
active in the mitochondrial system. The latter
compounds were tested at concentrations ranging
from 1 X 10-4 m to 6 X 10-* and reacted readily
with DPNH in the presence of crystalline lactic
dehydrogenase. Lupine mitochondria in addition
will catalyze the oxidation of DPNH in the
presence of hydroxypyruvic acid. However, cin-
namic and phenylglyoxylie acids are inactive.
Phosphate buffer extracts of wheat seedlings and
of acetone powders prepared from lupine cotyle-
dons will also carry out the reaction between
phenylpyruvate and DPNH. (Supported in part
by a grant from the American Cancer Society.)

764. Ion exchange chromatography of human
hemoglobin. JEAN L. Cook ANd MartTIN
Morrison (introduced by W. 8S. McCann).
Depts. of Medicine and Biochemistry, Univ. of

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

235

Rochester School of Medicine and Dentistry,

Rochester, N. Y.

Chromatography of oxyhemoglobin on columns
of IRC-50 ion-exchange resin has proved to be a
highly sensitive method for the separation of the
hemoglobins of normal adults. This technique
has demonstrated 3 chromatographically distinct
components in hemoglobin from normal adults.
The method has now been applied to a study of
the hemoglobin occurring in the human at birth.
These studies indicate that cord blood contains
at least 3 different hemoglobin fractions. The
major fraction is the first eluted from the column
and comprises approximately 70% of the hemo-
globin of pooled cord bloods. The slowest moving
of the hemoglobin fractions comprises about 10%
of the total eluted hemoglobin. It is identical
chromatographically to a component present in
normal adult hemoglobin. The remainder moves
in the region of hemoglobin ‘A’ (the major fraction
of normal adult hemoglobin). A study was also
made of that portion of cord hemoglobin which
resisted denaturation at pH 12.7. The undenatured
hemoglobin retained the spectral characteristics
of oxyhemoglobin and was not precipitated by 4
saturated ammonium sulphate. Chromatography
of this alkaline-resistant hemoglobin revealed
that only the slowest moving fraction completely
resisted denaturation. The fetal hemoglobin
(major fraction in cord blood) was eluted more
slowly from the column after alkali treatment.
Studies of this change in behavior are in progress.

765. ATP-ase and P**-exchange reactions in
the phosphorylating enzyme complex from
mitochondria. CectL CooPpEeR* AND ALBERT L.
LEHNINGER. Dept. of Physiological Chemistry,
Johns Hopkins Univ. School of Medicine, Balti-
more, Md.

A lipoprotein enzyme complex catalyzing
electron transport and coupled phosphorylation
has been separated from digitonin extracts of rat
liver mitochondria by differential ultracentrifuga-
tion. P:O ratios as high as 2.8 have been observed
for the DPN-linked oxidation of D-8-hydroxy-
butyrate by molecular oxygen. ADP is the specific
P acceptor; IDP, GDP, CDP and UDP are in-
active. Phosphorylation is uncoupled by DNP,
Dicumarol, gramicidin and pentachlorophenol
but not by Cat* or thyroxine. Mg*t* additions
uncouple phosphorylation. The preparations show
only slight ATP-ase activity in the absence of
added Mg**; this is greatly stimulated by DNP.
However, DNP does not evoke hydrolysis of
UTP; ITP, CTP; GTP, UDP; IDP, CDP’ and
GDP. No latent ATP-ase appears on aging of the
complex; rather there is loss of ability to respond
to DNP. The preparations catalyze incorporation
of labeled inorganic phosphate into ATP but not

236

into UTP, ITP, CTP, GTP nor the corresponding
diphosphates. The rate of the incorporation and
the final equilibrium reached are dependent on the
concentrations of P, ATP, and ADP and also the
pH. The characteristics of the P#-ATP exchange
reaction observed in the isolated enzyme complex
are somewhat different from those of intact
mitochondria, suggesting that intact mito-
chondria catalyze incorporation of P*? into ATP
through additional exchange reactions which are
not necessarily related to oxidative phosphoryla-
tion.

766. Effect of interference of methyl groups
on conformation of steroid side chain.
Joun W. Corcoran* ann H. HirscHMANN.
Depts. of Medicine and Biochemistry, Western
Reserve Univ., Cleveland, Ohio.

To explore the effect of in'eraction of the C-18
and C-21 methyl groups on stability, a rigid struc-
ture with a close approach of these groups was
obtained by the preparation of 36-acetoxy-168-
hydroxy-20-isobisnorallocholanic 22 — 16-lactone.
The lactone ring hydrolyzed slower than the one
of the 20-normal epimer which, however, was
found to be more stable in equilibration experi-
ments. The results indicate considerable hin-
drance to the free rotation of the steroid side
chain around the C-17-C-20 bond, but show
that even structures with considerable inter-
ference can be prepared and that these can persist
under strenuous conditions. Provided the stability
of both 20-epimers can be compared, the com-
pression effect can be used for configurational
assignments at C-20 if it is part of a small ring.
(Supported by Grant C-1679 from the Natl. Insts.
of Health, PHS.)

767. Lipide analysis of cellular fractions in
liver necrosis. W. E. CoRNATZER AND DUANE
G. Gatio.* Dept. of Biochemistry, Univ. of
North Dakota Med. School, Grand Forks.

Male albino rats of the Wistar strain were main-
tained on a stock diet containing 30% protein.
Acute liver necrosis was obtained in 12 rats within
48 hr. by the intraperitoneal injection of bromo-
benzene in corn oil using the method of Koch-
Weser (Proc. Soc. Exper. Biol. & Med. 79: 196,
1952). Eleven control animals received an injec-
tion of corn oil. Forty-eight hours later the livers
were rapidly removed and divided into two por-
tions, one for histological examination and the
other for lipide analysis. The composition of the
total-lipide phosphorus, lecithin phosphorus,
cephalin phosphorus and cholesterol was deter-
mined in mitochondria, nuclei and homogenate
preparations of the liver. Histological examina-
tions showed acute, central, fairly extensive,
often confluent necrosis in all liver preparations

FEDERATION PROCEEDINGS

Volume 1§

that were used. A statistically significant decreage
occurred in the total lipide, lecithin and cephalin
phosphorus in the homogenate fraction of the
necrotic livers as compared to the controls (P <
0.01) in the necrotic livers. A decrease was observed
only in the cephalin phosphorus of the liver
mitochondria fraction in necrosis. In the nuelei
of the necrotic livers a decrease in the lecithin
phosphorus (P < 0.05) and an increase in total
cholesterol (P < 0.01) were noted

768. Theobromine, theophylline and caffeine
metabolism in man. HERBERT H. Cornisn*
AND ApAM A. CurisTMAN. Dept. of Biological
Chemistry, Univ. of Michigan, Ann Arbor.

By a combination of column and paper chro-
matography, precipitation as silver salts and
ultraviolet spectrophotometry, the major meta-
bolic products in man of theobromine (3,7-di-
methylxanthine), theophylline (1,3-dimethyl-
xanthine) and caffeine (1,3,7-trimethylxanthine)
have been identified and quantitatively deter-
mined. A serial quantitative precipitation of the
silver salts of 7-methylxanthine and 3-methyl-
xanthine in the presence of theobromine was pos-
sible by adjustment of the pu first to 1.0 (for
7-methylxanthine) and then to a pH of 5.5 (for
3-methylxanthine). After the ingestion of 1 gm of
the di- or trimethylated xanthines by each of the
2 male subjects, the following average percentages
of the administered compound were found in
the urine: for theobromine, 28% as 7-methyl-
xanthine, 18% as 3-methylxanthine, 4% as 7-
methyluric acid and 12% as unchanged theo-
bromine; for theophylline, 35% as 1,3-dimethyl-
uric acid, 19% as 1-methyluric acid, 13% as 3-
methylxanthine and 10% as unchanged theo-
phylline; for caffeine, 27% as 1-methyluric acid,
9% as 1,3-dimethyluric acid, 19% as 1-methyl-
xanthine, 6% as 7-methylxanthine, 5% as 1,7-
dimethylxanthine and 1% as unchanged caffeine.
In man, theobromine and theophylline readily
lose one methyl group and caffeine two methyl
groups. The absence of any significant increase in
uric acid excretion suggests that the monomethyl-
xanthines are not demethylated to xanthine.

769. Function of carbonic anhydrase in the
alligator. Ronatp A. Counson AND THOMAS
HERNANDEZ. Dept. of Biochemistry, Louisiana
State Univ. School of Medicine, New Orleans.
Since carbonic anhydrase inhibition by 6068

(acetazoleamide) leads to a decrease in COQ:

and an increase in chlorides in alligator urine, it

appears that carbonic anhydrase is involved in

anion conservation. To test the influence of 6063

on the excretion of various cations and anions,

the following compounds were injected: NaCl,

Na2SO,, NaHCO; and NasHPO,. The excretion

ime 1§

crease
phalin
of the
(P<
served
liver
nuclei
cithin
- total

ffeine
‘NISH*
logical

chro-
3 and
meta-
|, 7-di-
ethyl-
thine)
deter-
of the
ethyl-
S pos-
) (for
> (for
gm of
of the
tages
nd in
athyl-

theo-
ethyl-

theo-
acid,
ethyl-
3 1,7-
feine.
sadily
ethyl
ase in
sthyl-

2» the
[OMAS
siana

CO;
ne, it
ed in
6063
1ions,
NaCl,

etion

=—

March 1956

pattern was;studied and compared with similar
experiments in which 6063 was given in addition
to each of the salts. In each case, urinary NH; was
decreased by Na, and CQ2 was decreased by the
various anions. SO, alone eliminated CO: from the
urine. Since there was no CO; in the urine of the
§0,-injected alligators, 6063 had no significant
effect on the anions in this experiment. Carbonic
anhydrase inhibition caused an increase in K in
all experiments and an increase in Cl in all but the
NasSO, group. In no case did 6063 significantly
affect the excretion of any anion other than Cl.
Carbonic anhydrase in the alligator normally
functions in the conservation of K and Cl. NH;
excretion was not related to carbonic anhydrase
activity. (Supported in part by Public Health
Service Grant No. H-2062.)

170. Metabolism of methyl histidine com-
pounds in animals. Ropert W. CowGILu AND
BERNARD FREEBERG (introduced by H. O. L.
FiscHER). Dept. of Biochemistry, Univ. of Cali-
fornia, Berkeley.
1-Methy]-histidine is a normal excretory product

of animals and occurs in muscle as anserine (6-

alanyl-1-methy]-histidine). 3-Methylhistidine has

more recently been found (TALLAN et al. J. Biol.

Chem. 206 : 825, 1954) in the urine of certain species.

Work of Harms and Winnick (Biochim. et biophys.

acta 15: 480, 1954) and of others has indicated that

the methyl group of 1-methyl-histidine cannot be
removed in the chick or the rat but that this com-
pound is incorporated into anserine in the chick.

In order to determine the utilization of 1-methyl-

histidine in a more direct fashion and to extend

this knowledge to 3-methyl-histidine, 1-C!4H;-

L-histidine and 3-C!4H;-histidine were synthesized.

These compounds were administered subcuta-

neously and intraperitoneally to rats, chicks,

rabbits and frogs. Most of the methylhistidine

compound was rapidly excreted unchanged (60%-

80% in 24 hr.) The resistance of these compounds

to degradation was demonstrated by the fact that

less than 1% of the radioactivity appeared in the
respired CO2 after either 1-methyl- or 3-methyl-
histidine were given. The products excreted and
those retained in the body were identified by
paper chromatography and by isolation with
unlabeled carriers. 1,3-Dimethyl-histidine [1,3-
dimethyl-4-(8-carboxyl-8-amino ethyl] imida-
zolium chloride) was considered as a possible
intermediate in the methylation and/or inter-
conversion of 1-methyl- and 3-methyl-histidines.
1,3-(C14H;)2-histidine was synthesized and in-
jected into animals of the above species. Excre-
tion of this compound was rapid and no radio-
activity appeared in the respired COs, which is
contrary to what might have been expected were

”:
ate haw

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

237

this compound converted to either 1-methyl- or
3-methyl-histidine in the animal.

771. Dissociation of nucleohistones. CHARLES
F. Crampton. Rockefeller Inst. for Med. Re-
search, New York City.

Evidence has been obtained that histones are
originally combined in calf thymus nucleohistone
in a manner which is different from that produced
when the components of nucleohistone are de-
liberately dissociated and then allowed to re-
associate. A distilled water solution of once-pre-
cipitated nucleohistone was prepared from calf
thymus under conditions not conducive to dis-
sociation (CRAMPTON, LIPSHITZ AND CHARGAFF.
J. Biol. Chem. 206: 499, 1954). A portion of this
nucleohistone was reprecipitated by raising the
NaCl concentration to 0.2 m. A second portion
was dissociated at 2 mM NaCl for 30 min., and its
components were reprecipitated at 0.2 m NaCl by
dilution with water. The two types of nucleo-
histone thus obtained, when maximally dissociated
by 0.6 m Ba(OAc)2-ethanol, yielded mixtures of
histones which were shown to contain similar
amounts of fractions A and B when chromato-
graphed on Ba IRC-50 (Crampton, Moore anp
Stein. J. Biol. Chem., 215: 787, 1955). However,
very different proportions of A and B were dis-
sociated from the two types of nucleohistone by
the action of Ba(OAc)2-ethanol at suboptimal Ba
molarity (0.15 or 0.2 m, pH 7-8). While the less
basic histone fraction A was bound least strongly
by the reassociated product, fraction A was
bound more strongly than the more basic fraction
B by the previously undissociated nucleohistone.
These observations were made using undialyzed
preparations. It has recently been found that
dialyzed, lyophilized preparations of calf thymus
histone occasionally contain a decreased propor-
tion of fraction B and an increased proportion of
material that moves through the column un-
retarded. Such variations have not been observed
in undialyzed preparations of histones dissociated
from rapidly prepared nucleohistone that has been
obtained by 1-2 hr. of extraction of the washed
tissue residue with distilled water instead of
the overnight extraction formerly performed.

772. Metabolism of p-methoxyphenylalanine
and p-methoxyphenylpyruvate. Dana I.
CRANDALL, JOEY PirruNG* AND LEONARD
GortrsMaN.* Dept.of Biological Chemistry, Univ.
of Cincinnatt College of Medicine, and Jewish
Hosp., Cincinnati, Ohio.

The experiments of Wakeman and Dakin (J.
Biol. Chem. 9: 139, 1911) in which pu-p-methoxy-
phenylalanine and p-methoxyphenylpyruvate were
shown to be ketogenic in perfused dog liver have
been reinvestigated. The procedures used by these

238 FEDERATION PROCEEDINGS

authors were followed except for 1) the use of
Nembutal instead of ether, 2) p- and L-p-methoxy-
phenylalanine were perfused individually, 3)
instead of running separate control experiments
the basal production of acetoacetate prior to the
addition of the test compound was measured
during the first 30 min. of each experiment and 4)
acetoacetate was determined by the method of
Walker (Biochem. J. 58: 699, 1954). Appreciable
quantities of acetoacetate were formed after ad-
dition of L-tyrosine (control experiment) to the
perfusion fluid and after p-p-methoxyphenyl-
alanine but not after L-p-methoxyphenylalanine.
Very small amounts accumulated after the addi-
tion of sodium p-methoxyphenylpyruvate. A non-
ketogenic p-amino acid (introduced as DL-serine)
gave rise to acetoacetate and exceptionally large
amounts accumulated following the addition of
ammonium L-p-methoxyphenylpyruvate. It seems
a possibility that the ketogenic action of DL-p-
methoxyphenylalanine and of p-methoxyphenyl-
pyruvate originally observed by Wakeman and
Dakin is due to the liberation of NH,*t by the
p-amino acid and to the use of ammonium salt of
the a-keto acid.

773. Experiments with C'!-labeled carbamyl
phosphate. R. Crokarrt,* M. E. Jones AND
F. Lipmann. Biochemical Research Lab., Massa-
chusetts General Hosp., and Dept. of Biological
Chemistry, Harvard Med. School, Boston.
C'4-labeled cyanate was prepared by melting

C'4-urea and carbonate. From this, radioactive

carbamyl phosphate (C!4AP) was prepared ac-

cording to Jones, Spector and Lipmann (J. Am.

Chem. Soc. 77: 819, 1955), using, however, 3 m of

phosphate/m of cyanate. This increases the yield

to 70% with regard to cyanate. The product was
either used without purification or purified through
barium precipitation from 25% methanol-water at
pu 8.5. The C'4AP was tested with extracts of
Streptoc8ccus faecalis R for reactions with aspara-
gine, glutamic acid, serine, threonine, glycine,
methionine, leucine, 8-alanine, proline, malate,
aspartic acid and ornithine. None except aspartic
acid and ornithine caused the appearance of
detectable new spots on the radioautograph of the
chromatogram. Furthermore, C!4AP was tested
with particle-free pigeon liver extract. The TCA-

filtrate was electrophorized at px 3.8 in 0.05 m

citrate and radioautographed. Two reaction

products were detected without addition, one with
added glutamine and two or more with added

ATP and ribose-5-phosphate, in addition to

the expected one with aspartate. The identifica-

tion of these various reaction products is now in
progress. (Supported by a grant from the Public

Health Service.)

Volume 15

774. Protection against teratogenic action of
azaserine and DON. C. P. Daag anp D. A,
Karnorsky (introduced by C. C. Stock). Div,
of Exptl. Chemotherapy, Sloan-Kettering Inst,
for Cancer Research, New York City.
O-diazoacetyl-l-serine (azaserine) and 6-diazo-

5-oxo-l-norleucine (DON) produce similar, yet

individually characteristic, developmental ab-
normalities in chick embryos. The effects studied
consist primarily of weight inhibition, eye lesions
and abnormal development of the beak, wings
and legs. The nature, severity and incidence of
defects depend upon the dosage and stage of
development at time of treatment; DON is ap-
proximately 50 times as active as azaserine by
weight. Since Hartman, Levenberg and Buchanan
(J. Am. Chem. Soc. 77: 501, 1955) reported that
azaserine interfered with the de novo synthesis of
inosinic acid in pigeon liver preparations, a series
of purines, nucleosides, nucleotides and meta-
bolically related compounds were tested for their
ability to prevent the appearance of the develop-
mental defects and growth inhibition produced
by azaserine and DON. Thus far, not all of the
compounds have been tested against both agents,
The chemicals were injected into the yolk sac of
4-day chick embryos shortly after yolk sac in-
jections of azaserine and DON. The embryos
were killed after the 12th day of incubation. The
following compounds protected the embryos
against azaserine and DON: adenine, adenosine,
hypoxanthine, inosine and 4-amino-5-imidazole-
carboxamide (AIC). The sodium salts of adeno-
sine-3-phosphoric acid, inosinic acid, and guanylic
acid protected against the effects of azaserine.

Guanosine and sodium guanylate protected against

DON. The following compounds did not prevent

the appearance of developmental defects in

embryos treated with azaserine: guanine, xan-
thine, uric acid, glutamine, serine, phenylalanine
and histidine. Glutamine was ineffective against

DON. Adenine also protected against azaserine

when injected 24 hr. after treatment with azaserine.

775. Binding of corticosteroids by plasma
proteins. Wiit1amM H. DavuGHADAY AND
CaroLyN Hartnetr (introduced by T. E.
WEICHSELBAUM). Dept. of Medicine, Washington
Univ. School of Medicine, St. Louis, Mo.
Binding of corticosteroids by plasma proteins

has been studied with dialysis equilibrium and

paper electrophoretic methods. Plasma from 2

patients receiving cortisone acetate and 1 patient

with Cushing’s syndrome was dialyzed against 4

volumes of phosphosaline buffer, pu 7.4. At equi-

librium more than 90% of the free 17-OH cortico-
steroids and between 47% and 66% of the 17-OH
corticosteroid-glucuronides were loosely bound to
plasma proteins. When human plasma was dialyzed

ple
do
do
fra

an
tid
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ori
aci
we
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Th
aci
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. Div,
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udied
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. The
bryos
osine,
1zole-
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inylie
erine.
rainst
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ts in
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anine
rainst
serine
erine.

asma

AND
Y. Bi
‘ngton

oteins
1 and
‘om 2
atient
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ind to
lyzed

March 1956

against 4 volumes of phosphosaline buffer con-
taining 0.5 ug/ml of hydrocortisone 4-C'*, or
corticosterone 4-C'*, between 65% and 75% of the
former and 86% of the latter were protein bound.
Human plasma fractions (Cohn) I, II, III, IVi,
IV, and V were dialyzed against hydrocortisone.
Significant binding occurred only with fractions
IV, and V. The binding by fraction IV,, however,
could be attributed to its contained albumin. The
association constant for hydrocortisone and
crystalline bovine serum albumin fell from 0.21 X
10-* to 0.12 K 10-4 with a rise of hydrocortisone
concentration from 0.7 to 452 um/1. At physiologic
concentrations of hydrocortisone the binding by 5
samples of human fraction V was consistently

higher (K = 0.40-0.64 X 10~‘) than by crystalline.

bovine serum albumin. Protein samples have been
dialyzed against buffers containing radioactive
corticosterone and hydrocortisone. At equilibrium,
filter paper was impregnated with the dialysate,
and protein solution was applied to the starting
line. After electrophoresis the binding of hydro-
cortisone was limited almost exclusively to the
albumin peak of human plasma and of human
fraction V.

776. Fractionation of ribonucleic acids from
yeast. FranK F. Davis* anp FRANK WortTH-
INGTON ALLEN. Dept. of Physiological Chemistry,
Univ. of California School of Medicine, Berkeley.
Ribonucleic acids from yeast prepared by the

method of Crestfield, Smith and Allen (J. Biol.

Chem. 216: 185, 1955) have been separated into 5

fractions by the use of salmine sulfate followed by

sodium dodecyl sulfate. At pu 6.5, 99% of the
ribonucleic acids is precipitated as salmine
tibonucleate. The salmine ribonucleate is only

10% soluble in 1-2 m sodium chloride but is com-

pletely dissociated in dilute solutions of sodium

dodecyl sulfate. Graded concentrations of sodium
dodecyl sulfate were used to fractionate. The
fractions which amounted to 91% of the original
material were analyzed for nucleotide composition
and subjected to ribonuclease action. The nucleo-
tide composition for the most readily soluble
fraction showed marked variations from the
original preparation in that guanylic and cytidylic
acids were high, and adenylic and uridylic acids
were low. The composition of the other fractions

did not vary greatly from the starting material.

The percentages of the cytidylic and uridylic

acids which were liberated as mononucleotides as a

result of ribonuclease action varied significantly

from fraction to fraction. No one of the fractions
produced the same quantity of free pyrimidine
mononucleotides as the original preparation
from which they were derived. Results indicate
that fractions from a given preparation of ribo-
nucleic acids may be defined more specifically in

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

239

terms of ribonuclease action than mononucleotide
composition. By way of contrast a dialyzed puri-
fied commercial sample of ribonucleic acids ex-
hibited entirely different solubility relationships.

777. Direct influence of hypoxia on iron ab-
sorption. JacK H. Davis anp C. A. JOHNSON
(introduced by Oar Brercerm). Dept. of Bio-
logical Chemistry, Univ. of Illinois College of
Medicine, Chicago.

Using the intestinal loop technique in white
male rats, experiments were designed to determine
iron absorption at reduced oxygen tension. Ab-
sorption was determined from the difference
between the iron injected into the loops and that
recovered after 6 hr., corrected for the inherent
iron content of the loops. No significant absorp-
tion of iron was observed in 6 hr. after 250 ug of
iron as FeSO, was injected into the loops of
normal rats. An average of 29 wg of iron was ab-
sorbed in animals made anemic by phlebotomy 6
hr. prior to injection. Animals placed in a de-
compression chamber at a simulated altitude of
25,000 ft. showed an average absorption of 54 ug.
When a gas mixture containing 4% CO: was used
to ventilate the chamber at simulated altitude,
iron absorption was reduced as compared to
animals breathing ambient air at reduced pres-
sure. Presumably, the presence of 4% CO: in the
chamber reduced the anoxemia in the animals by
directly stimulating the respiratory centers, the
decreased hypoxia accounting for reduced absorp-
tion. Increasing the oxygen content without
altering total pressure at simulated altitude was
not accompanied by the absorption of a significant
amount of iron from the loops. When a decrease in
oxygen supply to the loop was produced surgically,
absorption was enhanced. Adrenal insufficiency
had no effect on iron absorption. These studies
provide evidence that a local oxygen deficit in the
intestinal loop, regardless of etiology, acts di-
rectly to increase iron absorption.

778. Conversion of glycine to serine in extracts
of acetone-dried luminous bacteria. J.
WENDALL Davis,* JANET V. PASSONNEAU* AND
Joun R. Tortrer. Biology Div., Oak Ridge Natl.
Lab., Oak Ridge, Tenn.

Dowex-l-treated, water extracts of acetone
powders of a luminous Coccobacillus (Gest strain)
(CORMIER AND STREHLER. J. Cell. Comp. Physiol.
44: 277, 1954) contain enzymes for the conversion
of C14-glycine to C14-serine. Cofactors for this
C, transfer are tetrahydrofolic acid (THFA) and
pyridoxal phosphate. Folic acid with or without
DPN-H was unable to substitute for THFA and,
unlike the system found in Clostridium (WricuHT.
Federation Proc. 14: 308, 1955), citrovorum factor
is also inactive. Overnight dialysis of the extract

240

did not incite the need of additional cofactors.
The ability of both aged and dialyzed extracts to
incorporate glycine 2-C'4 could be enhanced
through the addition of 2,3-dimercaptopropanol
(BAL). Formaldehyde and the 8-carbon of serine
served as sources of the C, unit. In contrast to the
system found in pigeon liver (KisLiuK AND
Saxami. J. Am. Chem. Soc. 76: 1456, 1954), formate
was not incorporated into serine, even in the
presence of DPN-H and ATP. Methanol could not
be utilized in this transfer. The concentration of
THFA necessary for optimum incorporation of
label into serine bears a 1:1 molar relation with
the concentration of unlabeled serine added at the
beginning of the incubation as a C, pool. Both
DPN and DPN-H were inhibitory to the incorpo-
ration of glycine label into serine when unlabeled
serine was used as the C,; donor.

779. By» Determination and application of
serum vitamin binding capacity. RoBert L.
Davis,* Ropert C. Duvati* anp Bacon F.
Cuow. VA Hosp. and Dept. of Biochemistry,
School of Hygiene and Public Health, Johns Hop-
kins Univ., Baltimore, Md.

A rapid, simple and quantitative method for
the measurement of the capacity of human serum
for binding cobalt™-tagged vitamin Bi2 presented
earlier (Federation Proc. 13: 1954) was further
improved. This method is based on the finding
that the ‘resting’ cells of Lactobacillus leichmannii
ATCC 4797 can adsorb only the unbound vitamin
By from the serum-vitamin mixture. Using non-
sterile technique, an analysis of the data indicated
reproducibility. (S.D.+5%). This paper is con-
cerned with two applications of this method:
First, the investigation of the serum vitamin Bi:
(cyanocobalamin) binding capacity of tuber-
culous patients with and without complicating
diseases. Some of the findings include: a) deter-
minations of several hundred vitamin Bj: binding
capacitigs on serum obtained from patients with
minimal to far advanced pulmonary tuberculosis
demonstrated that such sera can bind from none
to all of the added radiovitamin at the 4-m yg
level; b) the serum vitamin By. binding capacities
of tuberculous patients with virus hepatitis were
less than 10% of the added vitamin during the
acute stage of liver disease but rose to approxi-
mately 50% with convalescence. Second, a com-
parative study of the capacity of tuberculous
serum samples to bind several analogous cobala-
mins. It was found that these analogues (hydroxo-,
chloro-, nitro- and sulfato-) did not differ from
cyanocobalamin in their binding properties.

780. Distribution and retention of cadmium
115 in the albino rat. CLARENCE F. DEcKER*
AND RicHarD U. Byerrum. Kedzie Chemical
Lab., Michigan State Univ., East Lansing.

FEDERATION PROCEEDINGS

Volume 1§

A comparison of the spatial and temporal
distribution of orally and intravenously ad-
ministered high specific activity cadmium 11§
has been made in the albino rat. In one experi-
ment 10 adult male rats were administered ap-
proximately 2 mg of cadmium 115 (as CdNOs) by
stomach tube. Two animals were killed at 8, 24,
72, 180 and 360 hr. after administration of the
radioactive cadmium. At the end of 72 hr. over
95% of the radioactive cadmium was found to
have been excreted in the feces. Less than 3%
of the administered cadmium was absorbed, and
the liver and kidney each contained a little over
1% of the original dose. Little or no activity was
found in the spleen, adrenals, bone and urine,
In a second experiment 20 adult male rats were
injected intravenously with approximately 280ug
of cadmium 115. Four rats were killed at 4, 8, 24
and 72 hr. and 1, 2 and 6 wk. after administration
of the cadmium. Plasma, kidney, liver, bone,
feces and urine were analyzed for cadmium 115
content. The largest percentage of the injected
dose was found in the liver, gut, kidney and feces.
Approximately 20% of the injected dose was ex-
creted by way of the gut at the end of 72 hr. Little
or no radioactivity was found in the urine or
bone at any of the time intervals studied.

781. Tissue cadmium content following ad-
ministration of low concentrations of
cadmium in drinking water. Lucite E,
DeEcKER (introduced by Rospert M. Hersst),
Kedzie Chemistry Lab., Michigan State Univ.,
East Lansing.

Male and female albino rats were given, ad
libitum, drinking water which contained either
0.1, 0.5, 2.5, 5.0 or 10 ppm cadmium (as cadmium
chloride). Control rats drank distilled water.
All the animals were fed the same diet. Rats were
killed at the end of 6 months, or at the end of 1 yr.
Liver and kidney tissues were analyzed for cad-
mium by the micromethod of Saltzman (Anal.
Chem. 25: 493, 1953). After 6 months, tissues from
rats drinking 0.1 ppm contained no cadmium, but
after 1 yr. there were about 1.5 ug cadmium in
kidney and 0.2 wg cadmium in liver/gm. fresh
tissue weight. Both tissues from rats drinking 10
ppm contained 15-25 ug cadmium/gm at 6 months,
whereas at the end of 1 yr. there were approxi-
mately 30 ug and 70 ug cadmium/gm of liver and
kidney, respectively. Generally, there was a much
greater concentration of cadmium (per gm fresh
tissue weight) in the kidney than in the liver. At
the end of 1 yr., between 2 and 4 times the amount
of cadmium was found in both tissues as was
present at the end of the 6-month period.

782. Isolation from ‘acerola’ juice of a fraction
containing bound ascorbic acid. ALICE DEL
CaMPILLo* AND Conrapo F. AsENso. Dept. of

con
0.2
pre
ase
heii

783.

N)
cres
D;
citr
that
tion
rats
of n
ihe
citr:
tion

Witl

me 1§

poral

ad-
1 115
peri-
1 ap-
3) by
3, 24,
f the
over
id to
1 3%
, and
over
r was
rine,
were
280ug
8, 24
ation
pone,
a 115
acted
eces.
S$ ex-
ittle
1e OF

- ad-
s of
BE,
BST).
/niv.,

1, ad
‘ither
nium
rater.

ction
E DEL
pt. of

March 1956

Biochemistry and Nutrition, School of Medicine,

Univ. of Puerto Rico, San Juan.

Ascorbic acid not extracted by ethanol, but
which is liberated after acid hydrolysis, is defined
as bound ascorbic acid. Its presence has been re-
ported by several investigators, both in plant and
animal tissue. We have separated from acerola
juice (Malpighia punicifolia L.) a solid fraction
containing bound ascorbic acid. It was precipi-
tated out of the juice on the addition of 3 volumes
of ethanol. The precipitate thus obtained was
washed repeatedly with ice-cold ethanol, under a
CO. atmosphere, until no free ascorbic acid could
be extracted. The dried precipitate has a grayish-
brown color, is amorphous and very stable if kept
in a desiccator. Although it contains 1-1.5% N it
gave a negative biuret, ninhydrin and Millon’s
tests. It does not contain sulfur or halogens. The
Molisch test was positive but the starch-iodine
test negative. When this fraction was hydrolyzed
with dilute metaphosphoric acid it yielded as-
corbic acid which was characterized by comparing
the absorption spectrum curve of the H2S0Q,-
osazone complex with one prepared with pure
ascorbic acid. The yield of the amorphous fraction
containing bound ascorbic acid was of the order of
0.21% by weight of juice. The bound ascorbic acid
present in the fraction represents 0.2% of the total
ascorbic acid in the juice. (Supported by a Guggen-
heim Fndn. Grant.)

783. Vitamin D and citrate oxidation. HEcToR
F. De Luca,* Freprik C. Gran* aND HARRY
SrrenBock. Dept. of Biochemistry, Univ. of
Wisconsin, Madison.

Since urinary citrate and tissue citrate are in-
creased in rats by the administration of vitamin
D, an investigation of citrate metabolism via the
citric acid cycle was undertaken. It was found
that vitamin D markedly reduced citrate oxida-
tion in homogenates of kidney tissue taken from
rats kept on either rachitogenic diets or on diets
of normal Ca and P content. It had no effect on
ihe oxidation of oxalacetate and consequent
citrate accumulation. The effect on citrate oxida-
tion could not be accounted for by the increase in
kidney calcium in the vitamin D-fed animals.
With liver tissue the decrease in citrate oxidation
was not consistently demonstrable which har-
monizes with the failure to demonstrate citrate
accumulation consistently in this organ.

184. Biological synthesis of sulfuric acid
esters of steroid hormones or their metab-
olites. R. H. De Mero, Curistinge LewycKa*
AND MartTHa WIZERKANIUK.* Dept. of Bio-
chemistry, Jefferson Med. College, Philadelphia,
Pa.

A soluble preparation (‘microsome-free’ solu-
tion) from rat or beef liver or a fraction obtained

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

241

from it (precipitating between 1.7 and 2.3 m
ammonium sulfate) synthesizes sulfates of phenols
with the sole requirement of ATP, Mg** and
SO,". The presence of 2 enzymes participating in
the synthesis has been demonstrated. One of them,
the ‘activating’ enzyme, contributes to the forma-
tion of an ‘active sulfate’ intermediate. The other,
transferring enzyme, transfers the sulfate from
the intermediate to the substrate. The same type
of preparations produce the binding of dehydro-
epiandrosterone, estrone and estradiol. A drop of
60—70% in the bound steroid is observed in ab-
sence of sulfate. This indicates that at least to
that extent the steroids may be bound to sulfuric
acid. The requirements for this synthesis appear
to be the same as that of the sulfuric acid esters
of phenols. The enzymatic preparations used do
not form the glucuronides of phenols, even in
presence of glucuronic acid. (Aided in part by
Contract Nonr 229 (00) with the Office of Naval
Research and by Research Grants G-3839 and
A-740 (C2) from the Natl. Insts. of Health, PHS.)

785. Leucyl-AMP as an intermediate in the
amino acid dependent exchange between
P#2P%2 and ATP. J. A. DeMoss,* Saunt M.
Genutu* anp G. Davin Nove... Dept. of
Microbiology, Western Reserve Univ. School of
Medicine, Cleveland, Ohio.

Enzyme systems catalyzing an amino acid-de-
pendent exchange between labeled pyrophosphate
and ATP have been described in rat liver by
Hoagland (Biochim. et Biophys. Acta 16: 288, 1955),
in yeast by Berg (J. Am. Chem. Soc. 77: 3163, 1955)
and in a variety of microbial extracts by DeMoss
and Novelli (Biochim. et Biophys. Acta 18: 592,
1955). The enzyme system(s) from Escherichia
coli has been purified by conventional procedures
about 20-fold, using leucine as substrate. The
enzyme system exhibits a high degree of specificity
for the t-forms of the active amino acids and for
ATP. Other nucleotides like ITP, CTP, GTP and
UTP are inactive. Careful balance studies have
failed to reveal accumulation of intermediates or
net changesin concentration of reactants. Thissug-
gests an interaction between ATP and amino acid
resulting in removal of PP and formation of
aminoacyl-AMP on an enzyme surface. Leucyl-
AMP has been synthesized by treating the disilver
salt of AMP with the acid chloride of leucine and
the product purified by ion exchange chromatog-
raphy. The product is characterized by containing
only bound AMP which is liberated by treatment
with NH:OH in neutral solution with the corre-
sponding formation of leucine hydroxamate.
Incubation of leucyl-AMP and P#P* with the
purified enzyme system results in formation of
ATP®, characterized by chromatography, by
hexokinase assay and by specific radioactivity.

242

These findings suggest that amino-substituted
acyl-AMP compounds are intermediates in the
exchange system.

786. Comparative study of plasma proteins of
amphibians and reptiles. HERBERT C. DEs-
SAUER AND WabDE Fox (introduced by F. G.
Brazpa). Depts. of Biochemistry and Anatomy,
Louisiana State Univ. School of Medicine, New
Orleans.

Approximately 600 specimens of 95 kinds of
amphibians and reptiles were collected or obtained
from scientists in other sections of the country.
Paper electrophoretic patterns of plasmas from
these animals were determined with an LKB
instrument (barbital buffer px 8.6, ionic strength
0.05, 15% glycerol). Samples were run 17 hr. at
170 v. and 4 ma. A survey of about 1000 patterns
indicates that the number of well separated pro-
tein components, as well as their concentration
and mobility, is characteristic of a particular kind
of animal. Very distinct pattern differences at the
subspecific level of taxonomy have been noted in
plasma from king snakes, water snakes and racers.
At higher taxonomic levels it is possible to dif-
ferentiate between frogs, salamanders, croco-
dilians, turtles, lizards and snakes by major
pattern characteristics. Plasma from young speci-
mens either contain in lower concentration or
lack one or more of the slower moving fractions
seen in patterns of adults. Sex differences occur in
plasma patterns of both amphibians and reptiles.
In females of at least 2 genera of viviparous
snakes, the season of follicular growth is accom-
panied by a great increase in one fraction of
plasma protein.

787. Spectrophotometric determination of
FAD. ALrrep DeutTscH AND RoBERT LAZZARINI
(introduced by Wrii1amM Drei). California
Fndn. for Biochemical Research, Los Angeles.
FAD can be assayed by utilizing a pair of enzy-

matic feactions. p-aspartic acid oxidase in the

presence of its coenzyme, FAD, converts p-as-
partic acid to oxalacetic acid and the rate of the
reaction is dependent upon the FAD concentra-
tion provided there is an excess of substrate and
apoenzyme. Catalase removes the peroxide
formed. The oxalacetic acid, in the presence of
malic dehydrogenase and reduced DPN, is con-
verted to malic acid while a stoichiometric amount
of oxidized DPN is formed. The rate of DPN
oxidation is readily observed by following the de-
crease in optical density at 340 my. As little as

0.2 ug of FAD/3 ml assay volume may be deter-

mined accurately. The same system may be used

for the assay of p-aspartic acid. Since the oxidase
acts on D-glutamic acid as well as p-aspartic acid

(Strut and Speruina. J. Biol. Chem. 182: 585,

1950), both can be determined by the addition of

FEDERATION PROCEEDINGS

Volume ij

glutamic acid-oxalacetic acid transaminase and
L-aspartic acid to the system. In this case, a-keto.
glutaric acid formed from p-glutamic acid reaets
with L-aspartic acid to yield the stoichiometric
amount of oxalacetic acid which is quantitatively
measured as described above.

788. Some properties of a hyaluronic acid
from human serum. H. F. Derutscu yp
JANE I. Morton. Dept. of Physiological Chem-
istry, Univ. of Wisconsin, Madison.

A marked precipitate was found to form upon
acidification of the serum of a cancer patient,
This serum possessed an electrophoretic com.
ponent migrating ahead of albumin at neutral and
slightly alkaline pH values which constituted from
1-1.5% of the total refractive index increment. In
the ultracentrifuge a component of sedimentation
constant lower than serum albumin was noted. 4
small amount of the substance was purified by a
combination of salt fractionation and electrical
transport techniques. The material gave no ab-
sorption maxima at 280 or 260 my but showed an
increasing absorption at the lower wavelengths. A
hydrolysate of the material tested negative for
a-amino acids. The sample assayed near 34%
hexoseamine. Its properties are strongly modified
by treating with hyaluronidase and the material
thus appears to be hyaluronic acid. A single com-

ponent was evidenced upon electrophoretic study |
and exhibited anionic properties even at px 1.5. |

The shape of the electrophoretic mobility curve
suggested an acidic grouping with a pK near 26.
The sedimentation behavior of the single com-
ponent was strongly concentration dependent
giving a value at infinite dilution near 2.4 S. The
diffusion constant was 2.8 X 1077 cm.? sec.“
Using a partial specific volume of 0.65 as reported
for hyaluronic acid a molecular weight near 56,00
is calculated. From the above data an axial ratio
between 50 and 60 may be calculated. Other
properties of this hyaluronic acid are noted.

789. Stimulation of mouse pancreas enzyme
secretion and respiration in vitro by drugs
and pancreozymin. SHERMAN R. DICKMAN
AND GENE A. Morritu. Dept. of Biological
Chemistry, Univ. of Utah College of Medicine,
Salt Lake City.

When mouse pancreas is incubated in Krebs
buffer for 2 hr. the zymogen granules remain in the
tissue, and approximately 5% of the total activity
of ribonuclease (RNase) or amylase is found in the
medium. The presence of the drugs pilocarpine or
carbamyl] choline, or a preparation of the hormone
pancreozymin, markedly increases the amounts of
either RNase or amylase which passes into the
medium under these conditions. Approximately
25% of the total RNase is found in the medium at
a final concentration of 5 X 10-7 m pilocarpine,

bat

the
diti
tric

lose
Nal
tris
pro
bies
and
app
Inte
Nat
Asi
whi
chai
stea
alte
cont
vest

791.

plume 1§

ase and
a-keto-
1 reacts
ometrie
tatively

ic acid
CH AND
Ll Chem-

mM upon
patient,
ic com.
tral and
ed from
nent, In
ntation
oted. A
ed by a
ectrical
no ab-
ywed an
igths. A
tive for
ar 34%
nodified
naterial
le com-
c study
pH 1.5.
y curve
ear 2.6,
le com-
pendent
-S. The

sec.
eported
r 56,000
al ratio
Other
dd.

nzyme
- drugs
ICKMAN
‘ological
edicine,

. Krebs
n in the
activity
d in the
rpine or
ormone
punts of
nto the
imately
dium at
carpine,

March 1956

1 X 10°: M carbamylcholine or 0.10 mg% of
pancreozymin. This concentration of pancreo-
zymin stimulates amylase secretion to an even
greater extent—35% of the total activity is found
in the medium. Graded responses in enzyme
secretion are obtained by varying the pancreo-
zymin concentration. Incubation of pancreas
under anaerobic conditions prevents enzyme
secretion in the presence of either pilocarpine or
pancreozymin. The chromatographic elution pat-
tern of the secreted RNase will be compared to
that which remains in the tissue. These 3 agents
likewise increase the rate of pancreas respiration.
The Qo2 is increased in the range 20 to 40% over
the controls at the above-stated concentrations.
Secretin also enhances Qo, but does not stimulate
enzyme secretion.

7%. Alternative pathway of the ribose-5-
phosphate metabolism in human red blood
cells. ZACHARIAS DiscHE AND Haroutp Sut-
GEuRA.* Dept. of Biochemistry, College of Physi-
cians and Surgeons, Columbia Univ., New York
City.

Ribose-5-phosphate (R5P) added to a dialyzed
fluoridate human hemolysate adjusted with tris-
buffer to pH 8.4 partially isomerizes reversibly at
0.01 mw and 33°C in 6 min. to ribulose- and xylulose-
5-phosphate and later reversibly breaks down to
a mixture of glucose, fructose-6-phosphate, sedo-
heptulosephosphate, triose-phosphate and fruc-
tose diphosphate identified enzymatically and by
paper chromatography with 3 solvents. If prior
to addition of substrate the hemolysate is incu-
bated for 4 hr. at 43°C, its transaldolase is in-
activated, no more hexose-6-phosphate is formed,
the breakdown of R5P yields under these con-
ditions equivalent amounts of sedoheptulose- and
triose-phosphate and all pentose which has disap-
peared is accounted for by the sum of sedoheptu-
lose, triose and fructose from FDP. If 0.1 m
NaHCO; is added prior to pH adjustment with
tris, the breakdown of R5P after inactivation
proceeds at an equal or smaller rate than without
bicarbonate, but the sum of sedoheptulose, triose
and fructose accounts for only about 70% of dis-
appearing pentose. Thirty per cent is transformed
into so far unidentified nonsaccharides; 0.1 m
NaCl is without effect on the breakdown of R5P.
A similar discrepancy between amounts of ribose
which disappeared and those recovered as sac-
charides appears if muscle aldolase is added in-
stead of NaHCO;. The reaction products of the
ilternative pathway and the mechanism of its
control by NaHCO; and aldolase are under in-
vestigation.

91. Incorporation of serine-3-C™ into methi-
onine. V. M. Doctor,* J. B. TRUNNELL* AND

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

243

J. Awapara. Univ. of Texas, M. D. Anderson

Hosp. and Tumor Inst., Houston.

Freshly prepared extracts of chick liver acetone
powder catalyze the incorporation of C'* derived
from serine-3-C"* into citrovorum factor (CF) and
methionine. It is necessary to add pteroylglutamic
acid (PGA), homocysteine, Mg**+, DPN and ATP
(Federation Proc. 14: 204, 1955). The enzymatically
labeled CF exhibits properties which are identical
to Leucovorin (Lederle). The labeled methionine
was identified as follows: 1) proteins were precipi-
tated from the incubation mixture with ethanol,
2) the alcoholic extract was desalted using ion-
exchange resins and 3) the desalted material was
chromatographed on paper with carrier methi-
onine. The carrier and the labeled compound gave
spots that were identical. Sufficient radioactive
unknown material was isolated using paper chro-
matography. The radioactive unknown material
was mixed with carrier methionine and recrystal-
lized twice. The material gave a count of over
4000. Other radioactive components were present
in the original incubation mixture as shown by
paper chromatography. One of them was an amino
compound (positive ninhydrin) and, using a pro-
cedure similar to that used to identify methi-
onine, the component was identified as cystathi-
onine. Additional, but not completed, studies are
being carried out to identify the other labeled
components.

792. Purification of relaxin by selective ion-
exchange chromatography. JoHN Doczi,
Greorce FE. Puitirps aND SyLvrA MALAMENT
(introduced by G. H. Maneun). Biochemistry
Dept. Warner-Chilcott Research Labs., Morris
Plains, N. J.

Purification of relaxin—the agent responsible for
pelvic relaxation in the estrogen-primed guinea
pig—was found possible by chromatography on
ion-exchange resin IRC-50. The method developed
takes advantage of the high iso-electric point of
relaxin (>7) and the fact that IRC-50 can be
buffered in this range to act as a selective absorb-
ent. According to results obtained, effluents of
relaxin solutions passed through IRC-50 columns
buffered at px 7, 8 and 9, contain 12, 32 and 100%
of influent activity, respectively, indicating maxi-
mum absorption in the pH range of 7-8. These
results suggest that the iso-electric point of relaxin
or a complex form thereof is in the neighborhood
of pH 8. The potency of the fraction eluted from
the ‘pH 7 column’ was 1830 guinea pig u/mg N.

793. Nutritional influences on conjugation of
bile acids by the rat. E. A. Dorsy, Jr., Marte
DanIELs* AND Sister M. Auicra ZIMMERMAN.*
Depts. of Internal Medicine and Biochemistry, St.
Louis Univ. School of Medicine, St. Louis, Mo,
To determine the effects of sulfur deficiency on

244

bile acid conjugation, rats in which bile fistulas
had been established were fed a purified ration
devoid of cystine and methionine. For investiga-
tion of the effects of pyridoxine on the conjugation
mechanism, the classical syndrome of Bg de-
ficiency was developed in the 2nd group of animals
prior to cannulation of the bile ducts. Bile samples,
after being freed of protein and extracted with
butanol, were submitted to reverse phase partition
chromatography according to the method of
Bergstrom et al. (Proc. Soc. Exper. Biol. & Med.
83: 71, 1953). Spectrophotometric analysis of the
eluates revealed, in agreement with previous
workers, that the normal rat secretes cholic acid
conjugated with taurine. As the deficiency states
became more severe, the amounts of taurocholic
acid in the bile decreased and increasing amounts
of a conjugate were detected in the glycocholic
acid zone of the Bergstrom chromatogram. De-
scending, one-dimensional paper chromatography
of the abnormal conjugate demonstrated that its
migration rates were identical with that of syn-
thetic glycocholic acid. Chromatography of the
hydrolysate from the glycocholic acid zones re-
vealed glycine; its migration rate and color with
ninhydrin were identical with that of authentic
glycine. On repletion of the deficient rats with a
complete ration, the abnormal conjugate dis-
appeared from the bile and cholic acid was found
to be present as the taurine conjugate.

794. New pathways in oxidation of D-galac-
tose and of D-arabinose. M. Dovupororr, J.
De Ley,* N. J. PALLERONI* AND R. WEIMBERG.*
Dept. of Bacteriology, Univ. of California,
Berkeley.

The following reactions are catalyzed by cell-
free extracts of Pseudomonas saccharophila grown
with galactose as substrate: p-galactose is oxidized
with DPN to p-galactono-y-lactone; the lactone is
enzymatically hydrolyzed to p-galactonic acid;
p-gala€tonic acid is dehydrated to 2-keto, 3-deoxy-
p-galactonic acid. This compound has_ been
crystallized as the potassium salt and has been
synthesized chemically from metasaccharin.
2-keto, 3-deoxy-p-galactonic acid is phosphoryl-
ated with ATP and split to yield pyruvic acid and
glyceraldehyde-3-phosphate. 2-keto, 3-deoxy,
6-phosphogalactonic acid is a probable inter-
mediate. Synthetic 2-keto, 3-deoxy-gluconic acid
is not attacked. The following reactions are
catalyzed by cell-free extracts of P. saccharophila
grown with p-arabinose as substrate: p-arabinose
is oxidized with DPN to a compound which is
probably p-arabono-y-lactone. p-arabono-y-lac-
tone is enzymatically hydrolyzed to arabonic acid.
p-arabonic acid is dehydrated to 2-keto, 3-deoxy-
p-arabonic acid. The structure of this compound
was established both by oxidation with ceric

FEDERATION PROCEEDINGS

Volume i§

sulfate and by conversion to the corresponding
a-aminoacid, which was degraded with periodate,
2-keto, 3-deoxy-D-arabonate is oxidized with DPN
to yield pyruvic and glycolic acids. The carboxy|
group of pyruvate is derived from C;, of arabinose,
while the carbinol group of glycolic is derived from
Cs. No pyruvic acid is formed in the absence of
DPN. Experiments with intact cells indicate that
the above mechanisms are operative in vivo

795. In vitro studies on incorporation of C4.
formate into nucleic acids of rabbit bone
marrow following total-body x-irradiation,
Caru D. Dovatass* ann Paut L. Day. Dept, of
Biochemistry, Univ. of Arkansas School of Medi-
cine, Little Rock.

In spite of much interest in the effects of x-ir.
radiation on the synthesis of nucleic acids, little
if any work has been reported on the study of this
effect in vitro. We have studied the effects of a
single total-body dose of 1000 r of x-rays on the
uptake of C14-formate in both ribonucleic acid and
deoxyribonucleic acid in whole, washed bone mar-
row cell suspension and in homogenates of the
crude bone marrow. The marrow was taken at
30-45 min. postirradiation. It has been found that
the irradiation inhibits the uptake of C"4-formate
into both RNA and DNA to an extent of approxi-
mately 50% when whole cells are used. The in-
hibition is somewhat less in the case of homoge-
nates. The ratio of specific activities of the 2
nucleic acids in both the control and irradiated
animals is approximately 1:1. The effects of vari-
ous experimental condition imposed in vitro along
with data from parallel experiments on nucleie
acid precursors have been studied.

796. Improved method for quantitative de-
termination of phosphorus. R. L. Dryer,’
A. R. Tammes* anv J. I. Rout. Clin. Bio-
chemistry Lab., Dept. of Biochemistry, College of
Medicine, State Univ. of Iowa, Iowa City.

Most methods for the determination of ortho-
phosphates depend on the reduction of phospho-
molybdic acid to a blue pigment. Benzidine has
been used as a very sensitive qualitative test for
phosphomolybdates since it is oxidized to a blue
pigment. The resultant sum of optical densities is
therefore higher for a given amount of phosphorus
than that obtained with most commonly employed
reagents. However, benzidine is unsuitable for
quantitative photometry of solutions since the
color is unstable. The isomer 2, 4’-diaminobiphenyl
(diphenyline) also reduces phosphomolybdates in
a manner similar to benzidine, forming pigment
mixtures of somewhat lower optical densities but
with stability of a much higher order. A method
based on the use of diphenyline has been de-
veloped for the determination of the phosphorus

tic

lai

ca]

dur
sto.

plume If

ponding
riodate,
th DPN
‘arboxyl
abinose,
red from
sence of
ate that
0.28

of C4.
it bone
liation,
Dept. of
of Medi-

of x-ir.
ls, little
y of this
cts of a
3 on the
vcid and
ne mar-
; of the
aken at
ind that
formate
1pproxi-
The in-
Lomoge-
f the 2
‘adiated
of vari-
ro along
nuclei¢

ive de-
)RYER,”
n. Bio-
ollege of

’ ortho-
hospho-
line has
test for
» a blue
sities is
sphorus
nployed
ble for
nee the
iphenyl
lates in
vigment
ties but
method
een de-
sphorus

March 1956

content of lipid digests. A modification of the basic
procedure has also been developed for the estima-
tion of serum inorganic phosphorus suitable for use
with fingertip samples of blood. The Beer-Lambert
law is obeyed in the range of 0.25 ug to at least 10
ug. The absorbance of 5 wg of phosphorus in a final
volume of 5 ml is 0.140. (This investigation has
been supported by a grant from the Natl. Insts.
of Health (NIH B-841), PHS.)

791. Titrimetric evidence for existence of
several DNA configurations: console model
universal titrator. Epwarp L. DuGGAN AND
Vincent L. Stevens.* Dept. of Physiological
Chemistry, Univ. of California School of Medicine,
Berkeley.

The highly organized DNA structure appears
labile to acid and alkali treatments of short dura-
tion. An efficient recording titrator was developed,
capable of rapid and accurate potentiometric
titrations. We desired to measure the free func-
tional groups of DNA with minimal destruction of
hydrogen bonds. Titration of various DNA speci-
mens showed the importance of the ionic history
of the specimens. The titer from pu 4-8 of DNA in
water was 20 times greater than that given by a
similar sample dissolved in m KCl. If a sample is
dissolved in water and dry KCl is later added to
molar concentration, the titer is still 4 times that
of DNA dissolved directly in KCl. Titrations per-
formed at 60° show qualitatively similar results.
DNA titrations in water from pH 8 to 2.5 show the
large titer (pH 5-2.5) characteristic of many free
purine and pyrimidine amino groups. Titrations in
u KCI show little titer from px 8 to 3.5, with a
sudden release of titratable groups between px 3.5
and pu 2.5. High ionic strength may confer sta-
bility on the hydrogen-bonded DNA structure by
providing ion layers about the DNA phosphoryl
groups at the time of hydration, thus eliminating
undesired effects due to electrostatic repulsion.
DNA may undergo configurational distortion
during solution in distilled water, or during
storage in solutions of low ionic strength.

798. Correlation of cytopathogenesis with
poliomyelitis virus release. THretma H.
DUNNEBACKE (introduced by C. A. Kniaut).
Virus Lab., Univ. of California, Berkeley.
Cultures of HeLa, monkey kidney and human

fetal cells infected with poliomyelitis types I

(Mahoney), II (MEF-1) and III (Saukett) undergo

similar reactions listed as 4 stages of cytopatho-

genesis: stage J, early nuclear pyknosis, no obvious
cytoplasmic change; stage JT, advanced nuclear
pyknosis with its displacement to one side of the
cell, constriction of cytoplasm; stage IJ, forma-
tion of a light-staining lesion in the center of the
cell, nuclear staining material condensed into a

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

245

small mass, cell rounded; stage IV, cell disintegra-
tion. Assays of the maintenance media following
infection indicate that virus release occurred at a
time interval between the development of cyto-
pathogenic stages III and IV. Similar studies with
human amnion cells showed a different series of
cytopathogenic reactions: stage J, disappearance
of nucleoli, no obvious cytoplasmic changes;
stage II, disappearance of the nucleus as a discrete
staining entity, cytoplasm not obviously changed;
stage III, cytoplasmic constriction and granula-
tion; stage IV, cell disintegration. Assays of the
maintenance media after infection showed that the
release of virus occurred at some time interval
between stages III and IV. The observations were
made with the light microscope on fixed, hema-
toxylin-stained cover slip cultures.

799. Dimethylthetin-homocysteine methyl-
pherase. J. DurRELL* anp D. G. ANDERSON*
(introduced by S. Kaurman). Lab. of Cellular
Pharmacology, Natl. Inst. of Mental Health,
Natl. Insts. of Health, Bethesda, Md.
Dimethylthetin-homocysteine methylpherase

has been purified from acetone powder extracts of

horse liver by means of adsorption on and elution
from calcium phosphate gel, heat treatment and
ammonium sulfate fractionation. Preliminary data
from ultracentrifugation and electrophoresis sug-
gest that in the presence of GSH the purified
enzyme may be homogeneous. In the absence of

GSH the enzyme appears to polymerize reversibly.

Based on a molecular weight of 150,000 the turn-

over number (uM/uM/min.) is 1500. The enzyme

seems to be relatively specific with respect to its
substrates, dimethylthetin and t-homocysteine,

800. Aldosterone content of urine and ab-
deminal fluid in patients with cirrhosis of
the liver. Inc—e Dyrenrurtu.* C. H. Stacey*
AND ELEANOR H, Vennina. McGill Univ. Clinic,
Royal Victoria Hosp., Montreal, Canada.

The aldosterone excretion has been studied in
patients with cirrhosis of the liver. The average
value for healthy individuals is 3.5 + 1.5 ug/24 hr.
One case of biliary cirrhosis showed a normal
value. In 4 patients suffering from portal cirrhosis
with ascites the excretion of aldosterone ranged
from 3.2 to 30.2 ug/24 hr., the average value of 16
determinations being 18.1 ug/24 hr. In 1 patient
aldosterone excretion was determined on 3 sepa-
rate occasions before and after paracentesis. Each
time there was a decrease in the aldosterone out-
put which was followed by a gradual increase in
the excretion of this hormone until the next
paracentesis. The aldosterone content of the as-
citic fluid was 0.090-0.129 ug/100 ml. There was no
correlation between the amount of accumulated
fluid and its aldosterone concentration. In a

246

woman with gross ascites the urinary aldosterone
was normal and was not affected by the withdrawal
of abdominal fluid. However, there had been mas-
sive hematemesis with shock prior to the assays.
In this case the aldosterone content of the ascetic
fluid was found to be within the range of that re-
ported by Simpson and Tait for normal blood.

801. A strain-dependent influence of glycine
on salicylate-induced adrenal ascerbic
acid depletion in rats. CHARLES H. Eapss,
Jr. anD J. Stanton Kina, JR. (introduced by
Hersert P. Sarett). Dept. of Biochemistry,
Univ. of Tennessee, Memphis.

It has been found that glycine, when adminis-
tered with salicyclic acid (SAL) to Wistar rats,
potentiates the action of SAL in decreasing the
adrenal ascorbic acid (AAA), although glycine has
no such effect alone. In this study, the effects of
glycine and n-acetylglycine (NAG), administered
with SAL, on the AAA depletion in Wistar and
Tennessee Medical Units (TMU) Colony rats are
compared. Results showed that, in contrast to the
effect of glycine with SAL in Wistar rats, there was
no evidence of any potentiating effect in the TMU
rats. NAG, when used in place of glycine, pro-
duced no significant effect on the action of SAL in
Wistar rats whereas, in the TMU rats, a significant
inhibition of the AAA depletion by SAL to the
extent of 39% was observed. However, when a
higher ratio (300 mg/kg b. wt. instead of 150
mg/kg) of SAL to NAG (300 mg/kg) was employed
in the injection mixture, the inhibition was re-
lieved correspondingly in the TMU rats. Several
other compounds selected for their relationship to
glycine as possible metabolic intermediates, either
utilizing glycine or yielding glycine, were tested.
None of these compounds produced any significant
enhancement of AAA depletion beyond that of
SAL alone.

802. Synthesis of 19-hydroxyandrostenedione.
MAXIMILIAN R. EHRENSTEIN AND Max DisnNEN-
BERGER.* Div. of Steroid Research, Dept. of Re-
search Medicine, Univ. of Pennsylvania, Phila-
delphia.

The synthesis, starting from strophanthidin, a
19-hydroxyprogesterone and 19-hydroxy-11-
desoxycorticosterone has been described. Evi-
dence in support of the normal configurations of
these compounds has been presented and the for-
mation of 19-hydroxyl substituted steroid hor-
mones by biological systems has been reviewed
(BARBER AND EHRENSTEIN. J. Organ. Chem. 19:
1758, 1954; 20: 1253, 1955). Thus, 19-hydroxy-11-
desoxycorticosterone, first synthesized in this
laboratory, has served for the identification of
material obtained from various biological sources
by several authors. Recently the formation of 19-

FEDERATION PROCEEDINGS

Volume if

hydroxy-A‘-androstene-3,17-dione (VII) by iney.
bating A‘-androstene-3, 17-dione with beef adrengl
homogenate was demonstrated (MreyiEr. Experiep.
tia 11: 99, 1955) and its significance as a metabolir
intermediate has been discussed (MEYER. Big.
chim. et Biophysiol. Acta 17: 441, 1944). A clear-cut
synthesis of this compound has now been achieved
by the following sequence of transformations:
38,19-diacetoxy-5-hydroxyetianic acid (I) (from
strophanthidin; Herzig and Ehrenstein, J. Organ,
Chem. 17: 713,’ 1952); 38,19 - diacetoxy - 21 - di.
azopregnan-5-ol-20-one (II) (m.p. 147-148.5°.
[a]> + 135°); 38,19-diacetoxypregnan-5-0l-20-one
(III) (m.p. 106-108°; [a]> + 88°); 38,5, 19-triace.
toxypregnan-20-one (IV) (m.p. 161.5-162°; [a]> +
75°); 38,5,178,19-tetraacetoxyetiocholane (V)
(m.p. 136-138°; [eJ + 11°); etiocholane-36,5,
178,19-tetrol (VI) (m.p. 211-212°; [a]> + 38%;
19-hydroxy-A‘-androstene-3,17-dione (VII) (mp,
172-173°; [a]>° + 188°; Amisx 242 my, €max 16,100);
19-acetoxy-A‘-androstene-3, 17-dione (VIII) (amor.
phous; Au’. 237 mu). The characteristics of VII
and VIII agree with those reported for the
compounds nbtained biologically (lit. cit.). (Aided
by research grants C757-C3 and C757-C4 from
the Natl. Insts. of Health, PHS.)

803. Changes in histochemical characteriza-
tion of skeletal muscle following disuse by
immobilization. LILLIAN EICHELBERGER AND
MicuaEt Roma.* Depts. of Surgery and Bio-
chemistry, Univ. of Chicago, Chicago, Il.
Disuse was brought about in a group of puppies

from the same litter by immobilization of one hind

leg of the puppy. The opposite leg being the
weight-bearing leg was used as the control leg. The
report given here covers the effects of this experi-
mental disuse on the histochemical characteriza-
tion of the skeletal muscle from both the calf
group and the thigh group of muscles. When 4
designated time had elapsed following immobiliza-
tion (6-8 wk. progressively) the puppies were
anesthetized with Nembutal and the calf and thigh
groups of muscles removed from both the control
and experimental legs for the analyses. All tissue
data were calculated on a fat-free basis. When the
analytical data for the muscles from the control
leg and from the immobilized leg were interpreted
histochemically and a quantitative comparison
made, the significant findings were: 1) a large in-
crease in the proportion of extracellular tissue,
ranging from 30 to 110%. The extracellular tissue
comprised both the extracellular fluid and the con-
nective tissue solids. The increase in weight of the
extracellular tissue seemed to correspond to the
duration of the immobilization; 2) a decrease in the

relative mass of muscle fibers of from 10 to 40%

without significant changes in the water, potas-

sium or magnesium concentrations in the muscle

ere
ch:

ad
an
ful
fac
ve
cos
an

vel
by
hy
nol

in|

sur
the

olume 5

by incu.
' adrenal
jx perien-
1etabolic
ER. Bio.
clear-cut
achieved
mations:
[) (from
T. Organ,
- 21 - di.
7-148.5°;
»1-20-one
9-triace-
; [als +
ne (V)
ne-38,5,
+ 38%),
I) (mp.
16,100);
[) (amor-
3 of VII
for the
). (Aided

>4 from

cteriza-
suse by
FER AND
nd Bio-

puppies
one hind
2ing the
leg. The
S experi-
acteriza-
the calf
When 3
nobiliza-
les were
nd thigh
» control
11 tissue
Vhen the
- control
erpreted
nparison
large in-
r tissue,
ar tissue
the con-
ht of the
1 to the
se in the
to 40%
, potas-
. muscle

March 1956

fibers. These. quantitative histochemical observa-
tions are consistent and supplement the histologi-
cal hypothesis that in atrophy of skeletal muscle
the internal structure of the muscle fibers does not
change. (Aided by Grant A-972, Natl. Inst. of
Arthritis and Metabolic Diseases, Natl. Insts. of
Health, PHS.)

904. Adrenocortical function in anesthetized
dogs during and after pyrogenic stress.
KristEN B. Erx-Nes (introduced by Leo T.
SAMUELS). Dept. of Biological Chemistry, Univ.
of Utah College of Medicine, Salt Lake City.
Numerous publications have reported an in-

creased secretion of ACTH during certain types of
change in body environment or ‘stress.’ Little is
known, however, about the functional state of
adrenocortical tissue in the acute phase of ‘stress’
and the ability of this tissue to regain proper
function in the period following ‘stress.’ These
facets of adrenocortical function have been in-
vestigated using levels of 17-hydroxycorti-
costeroids from the peripheral blood plasma of
anesthetized dogs given a pyrogenic dose of bac-
terial mucopolysaccharides (Piromen) intra-
venously. Following the administration of the
pyrogenic agent a sustained rise in plasma 17-
hydroxycorticosteroids was found. The intrave-
nous administration of a standard amount of
ACTH 4-6 hr. later resulted in an additional in-
crement in the already elevated levels of these
steroids. Since intravenously injected cortisol was
removed from the blood stream of the ‘stressed’
animal at a reduced rate during this period, the
rise after ACTH did not represent a large increase
in secretion. At least 2 factors are involved in the
apparent hypercorticism: increased production
and decreased catabolism. In the animals which
survive this ‘stress’ the standard ACTH test and
the standard cortisol removal test were performed
at 24-hr. intervals thereafter. During the first 3
days a reduced response was found. Normal
adrenocortical capacity was occasionally found as
early as the 4th day after ‘stress,’ and in all of the
animals, between the 6th and the 8th day. In
spite of the life-threatening ‘stress’ condition used,
the ability of the canine adrenocortical tissue to
elaborate 17-hydroxycorticosteroids in the pres-
ence of excess amounts of ACTH is not impaired
for a prolonged period of time.

805. Action of urethane on_ respiration,
growth and oxidative phosphorylation in
tetrahymena. JoHN J. EILer, Josepu Z.
KREZANOSKI* AND Kwan-Hva LeEg.* School of
Pharmacy, Univ. of California, San Francisco.
Cells of Tetrahymena geleii subcultured in 0.08 M

urethane grow definitely better than untrained

cells in concentrations of urethane up to 0.09 m.

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

247

However, both groups of cells are equally sensitive
to the respiration-depressing effects of the drug
and each shows a sharp break in the oxygen utili-
zation-drug concentration curve at 0.1 m. Clearly,
the growth-inhibiting effects of the drug which
occur at concentrations less than 0.1 m must be
due to other than inhibition of a special, sensitive
set of oxygen-consuming reactions as postulated
by Ormsbee and Fisher (J. Gen. Physiol. 27: 461,
1944). The effects of the drug on oxygen consump-
tion and phosphate uptake were studied on un-
washed mitochondrial preparations from trained
and untrained cells. The respiration of the mito-
chondria from the trained cells was about 70% of
that from the untrained cells. Both gave a P/O of
2.3-2.5 with a-ketoglutarate. In both, the respira-
tion increased with increasing urethane, reaching
a maximum at 0.2 m (40% increase) ; the P/O either
remained constant or increased slightly. At 0.3 m
urethane the respirations assumed the drug-free
level but the P/O dropped to about 1.2. Further
small increases in urethane depressed respiration
slightly and the P/O fell well below 1.0. Urethane
exerts an uncoupling effect almost identical to
that of dinitrophenol. Except for the concentra-
tions involved, there is much similarity between
the effect of urethane on the respiration of the
whole cell and on the mitochondria.

806. Enzymatic hydrolysis of glucuronolac-
tone. FRANK EISENBERG, JR. AND JAMES B.
Fieitp.* Natl. Inst. of Arthritis and Metabolic
Diseases, Natl. Insts. of Health, Bethesda, Md.
The failure of the whole animal to oxidize

glucuronate to CO», in contrast to the ready com-

bustion of glucuronolactone, has led to the belief
that these 2 closely related compounds are meta-
bolically distinct. Evidence will be presented at-
testing to the presence in rat liver of an enzyme
system that catalyzes the hydrolysis of glucurono-
y-lactone to glucuronate. Glucuronolactone dis-
appears rapidly when incubated at px 7.4 with rat
liver slices or homogenate. In the presence of
boiled tissue and in tissue-free controls, the com-
pound also disappears but at only 3 the rate. A
method has been developed for the determination
of glucuronate which consists of converting the
salt to the lactone followed by the hydroxamic
acid procedure for the lactone. By means of this
method and by acidimetric titration, an acid,
presumably glucuronic, was observed to form in an
amount equivalent to the lactone disappearing.
By paper chromatography of the reaction product
after incubation with uniformly labeled glucurono-
lactone-C* no radioactive compounds other than
the substrate and a spot corresponding to glu-
curonate could be detected. Isolation of the radio-
active product from the chromatogram and re-
peated recrystallization with carrier potassium

248

glucuronate established the compound as glu-
curonate. In a parallel experiment no CO. was
formed. With glucuronate as substrate no reversal
of hydrolysis was observed at px 7.4, 6.5 and 6.0.
At px 6.0 the enzymatic hydrolysis of the lactone
was completely inhibited. Brain, kidney, spleen,
diaphragm and testis showed no capacity to
hydrolyze glucuronolactone.

807. An 8-azaguanine-resistant strain of
Lactobacillus casei. GERTRUDE B. ELIoN,
SAMUEL SINGER* AND GEorRGE H. Hitcutnes.
Wellcome Research Labs., Tuckahoe, N.Y.

A strain of Lactobacillus casei has been isolated
which is 1000 times more resistant to 8-azaguanine
than is the wild strain. The resistant strain shows
a high degree of cross-resistance to 8-azaxanthine,
8-azahypoxanthine and 2,6-diaminopurine but not
to such purine antagonists as 6-mercaptopurine,
thioguanine, purine and 8-azaadenine. Reversal
studies show that, as in the wild strain, the in-
hibitory actions of 2,6-diaminopurine and purine
are reversed preferentially by adenine. However,
inhibiton by 8-azaadenine can be overcome by
either adenine or xanthine. The mutant grows well
in a folic acid-free medium containing thymine
and adenine or xanthine but its utilization of
hypoxanthine and guanine is poor. Its growth with
the purine nucleosides is also poor but guanylic
acid and adenosine-3’-phosphate support growth
well. Resistance to 8-azaguanine seems to be as-
sociated with the inability of the microorganism
to metabolize guanine and hypoxanthine. This
supports the generalization suggested earlier that
the elimination of the metabolic pathway affected
by the drug is a frequently used mechanism for the
development of drug resistance.

808. Comparison of two methods for intrinsic
factor evaluation. LEON ELLENBOGEN,* WIL-
uiaM L. Wiiuiams, S. Frep RABINER* AND
Hersert C. Licntman.* American Cyanamid
Co., Research Div., Pearl River, and Dept. of
Medicine, State Univ. of New York, New York
City.

Twenty-three intrinsic factor preparations of
varying potencies were assayed by both the classi-
cal clinical method using 35 pernicious anemia
patients in relapse and by an improved urinary
excretion test (U.E.T.) (ELLENBOGEN et al. Proc.
Soc. Exper. Biol. & Med. 89: 357, 1955) using 25
pernicious anemia patients in remission. The rela-
tionship between the amount of intrinsic factor
given each day in pernicious anemia in relapse and
the effect produced by the same amount in the
U.E.T. was investigated. With most of the in-
trinsic factor preparations, the minimum daily
dose to produce remission was determined in re-
lapsed patients. To obtain a daily excretion value

FEDERATION PROCEEDINGS

Volume 16

of about 7% (2 ug radioactive vitamin By» given)
an amount of intrinsic factor equivalent to 2 to3
daily oral doses was necessary. In only one in-
stance was the apparent minimum daily dose ip
pernicious anemia sufficient to give a satisfactory
excretion value. In the U.E.T. individual vari-
ation was minimized and the method made more
quantitative by use of 3 daily oral doses of g
thoroughly assayed intrinsic factor reference
sample with each patient. Agreement of the results
of the U.E.T. with those of the clinical method
indicated that the U.E.T. is a reliable method for
the evaluation of intrinsic factor preparations par-
ticularly when the proper dosage of intrinsic factor
is used in the latter test.

809. Oxidation of reduced pyridine nucleo-
tides in brain. SasHa ENGLARD* AND Haro
J. Srrecker. Albert Einstein College of Medicine,
Yeshiva Univ., and the New York State Psychiatric
Inst., New York City.

The metabolism of glucose appears to be the
most important if not the only source of the energy
required for the normal functioning of the nervous
system. The metabolic processes leading to the
production of energy from glucose oxidation are
known to be intimately involved with the oxida-
tion of reduced pyridine nucleotides. The oxida-
tion of DPNH and TPNH was studied with various
preparations of cerebral tissues of the rat, guinea
pig and ox. An aqueous extract cf an acetone
powder of beef brain cortex rapidly oxidized added
DPNH and TPNH in the presence of electron ac-
ceptors such as 2-methyl-1,4-naphthoquinone
(menadione), adrenochrome, methylene blue and
2,6-dichlorophenolindophenol. With equal concen-
trations of reduced pyridine nucleotides, in the
presence of menadione, the rate of oxidation of
TPNH was more rapid than that of DPNH. When
adrenochrome was substituted for medadione,
DPNH was oxidized more rapidly. The initial
oxidation rate of DPNH was inhibited more
strongly by dicumarol in the presence of
menadione than in the presence of adrenochrome,
whereas the inhibition of TPNH oxidation by di-
cumarol was greater with adrenochrome. Under
certain conditions with limiting menadione con-
centrations, the oxidation of DPNH proceeds
diphasically, which may indicate that the final
rate is determined by the autoxidation of reduced
menadione. This interaction of DPNH and mena-
dione is affected by the addition of cytochrome c.
Experiments under way may indicate the relation-
ship between these brain systems and similar
systems which have been reported in other tissues.

810. Kinetic and spectral studies of cyto-
chromes in DPNH oxidase. Ronatp Esta-
BROOK* AND Bruce MackteEr. Johnson Fndn. for

val

was

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Ext

me 16

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2 to3
ne in-
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uctory
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Esta-
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March 1956

Med. Phijsics, Univ. of Pennsylvania, Phila-
delphia and Inst. for Enzyme Research, Madison,
Wis.

The spectral and kinetic properties of prepa-
rations of ‘closed’ DPNH oxidase (Federation Proc.
14: 248, 1955) prepared from beef heart have been
investigated. The-difference spectrum (reduced
minus oxidized) of preparations at room tempera-
ture and the absolute spectrum of preparations at
—190°C show the presence of cytochromes a, as,
c,¢: and b. The relative content of the components
of the preparation is estimated to be 1: 0.7: 0.4:
0.5: 0.7 : 0.5 for cytochromes a, a3, c, ci, b, and
flavoprotein, respectively. Titration of the prepa-
rations of enzyme with DPNH in nearly stoichi-
ometric amounts has indicated that flavoprotein
and the cytochrome components account for about
70% of the reducing equivalents added. The
sequence of interaction of the cytochromes was
determined by studies of the influence of varying
concentrations of DPNH on the steady state
values of cytochrome components in an azide in-
hibited system. This showed that cytochromes
a+ a; attain maximal steady state reduction at
lower concentrations of DPNH than do cyto-
chromes ¢, c; and b, in that order. Kinetic studies
measuring the rate of reduction of the various
cytochromes have confirmed such a sequence. The
relative molar rates of reduction upon addition of
DPNH are 1: 3: 4: 30 for cytochromes (a + as),
¢c, c, and b. Turnover numbers and steady state
values of the cytochrome components of the prepa-
rations have been determined and agree with
values previously obtained by Chance for the suc-
cinoxidase system in Keilin and Hartree heart
muscle preparations.

811. Comparison of metabolites of hydro-
cortisone in normal and rheumatoid syno-
vial cavities. RIcHARD FAIRBANKS* AND HILDE-
GARD WiLson. Dept. of Medicine, New York
Univ. College of Medicine, New York City.

The possibility of a difference between normal
and inflamed synovial tissues in their actions on
adrenocortical hormones has been investigated.
We have previously described a pattern of metabo-
lites in pooled extracts of knee fluid from subjects
with rheumatoid arthritis and inflamed knee
joints, after hydrocortisone had been injected
intra-articularly (J. Clin. Endocrinol. & Metab.
Jan. 1956). In the present studies fluids from 5
theumatoid subjects were withdrawn one half
hour after the intra-articular injection of hydro-
cortisone free alcohol. The extract of each fluid
was chromatographed separately, using the tolu-
ene- and ligroin-propylene glycol systems. The 5
patterns of metabolites were almost identical with
each other and with that previously described.
Extracts of the synovial fluid of 2 normal knee

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

249

joints, obtained one half hour after injecting hy-
drocortisone, were also chromatographed. The
patterns of metabolites were identical, but differed
from those of the rheumatoid subjects. One of the
chief corticosteroid metabolites, found in 3 of the
5 rheumatoids, was absent in the controls. 118-
hydroxy-A‘-androstene-3,17-dione appeared pres-
ent in all 7 subjects. Both controls also showed a
less polar component whose properties indicated
it to be another C,,0; 17-ketosteroid. This was the
most abundant metabolite in the controls, but
was not detected in the rheumatoid fluids. It is
concluded that either the degradative metabolic
reactions, or the process of absorption through the
lining tissues was altered by the inflammatory
disease.

812. Enzyme inhibition by fluorocompounds.
ALAN S. Farruurst,* Ropert E. Smita* anp
Emery M. Gat. Depts. of Physiology and Phar-
macology, School of Medicine, Univ. of Cali-
fornia, Los Angeles.

Fluoropyruvate was shown to inhibit the oxida-
tion of pyruvate and of acetate in presence of
fumarate by rat liver mitochondria. It further in-
hibited the oxidation of 3-glycerophosphate, but
had. no effect on the oxidation of citrate or of
-ketoglutarate. Fluoropyruvate caused a decrease
in citrate formation from acetate and fumarate in
fluorocitrate-blocked rat liver mitochondria. Also,
it effected a decreased synthesis of fluorocitrate
from fluorcacetate and fumarate. In a phosphate-
free mitochondrial system fluoropyruvate ac-
celerated the release of inorganic phosphate from
ATP, using fumarate and pyruvate as substrates.
In a phosphorylating system containing inorganic
phosphate and fluoride, and with glucose and hexo-
kinase as acceptors, fluoropyruvate produced a
decrease in the P/O ratio, again with pyruvate
and fumarate. Fluoropyruvate, however, did not
produce a dinitrophenol-like effect on the oxida-
tion. Fluoromalate did not serve as substrate in
the TCA cycle to form fluorocitrate with acetate
and did not apparently inhibit the oxidation of
malate. Using indophenol as electron acceptor
with the pigeon breast pyruvic oxidase preparation
of Schweet, the oxidation of pyruvate was found
to be inhibited by fluoropyruvate, the inhibition
being apparently non-competitive, with no de-
carboxylation of the fluoropyruvate itself oc-
curring. Fluoropyruvate had no effect on the
cholinesterase of pigeon brain, but produced slight
inhibition of the pseudocholinesterase with
-methylacetylcholine as substrate. Fluoroacetate
and fluoroacetylhydroxamate were without effect
on these two enzymes.

813. Preparation and antimicrobial activity
of crystalline conalbumin. Rosert E.

250 FEDERATION PROCEEDINGS

FEENEY AND Marvin B. Ruopss.* Dept. of Bio-
chemistry and Nutrition, Univ. of Nebraska,
Lincoln.

Crystalline conalbumin prepared from egg white
according to Warner and Weber (J. Biol. Chem.
191: 173, 1951) was found contaminated with yellow
pigment and relatively large amounts of lysozyme.
The high lysozyme contents made such prepara-
tions unsuitable for our antimicrobial studies. In
addition, although crystalline iron complex was
easily obtained, only one preparation of the de-
ironed protein was crystallizable. The yellow pig-
ment has been removed by two reprecipitations at
pH 6.5 in app. 0.6 saturated ammonium sulfate.
This pigment had an absorption spectrum similar
to, but not identical with, riboflavin, and was non-
fluorescent. The lysozyme has been removed by
passing 3-6% aqueous solutions of either the iron-
free protein or the iron complex through ion-
exchange columns of IRC-50 resin buffered at
pH 6.7-7.2 with phosphate. Six preparations of
several times recrystallized iron-conalbumin
com>'*x had original lysozyme contents of 0.4-
2.1:, «wverage 1.1). After passage through the
resin, the lysozyme contents were reduced to
<0.001% (measured bacteriolytically). Similar
results were obtained with this purified crystalline
material as with our previously employed
amorphous material in studies of the general anti-
bacterial activity (FRAENKEL-CONRAT AND
Freeney, Arch. Biochem. Biophys. 29: 101, 1950),
and of the antagonistic relationships between
8-hydroxyquinoline and conalbumin (FEENEY.
Ibid, 34: 196, 1951). Organisms studied included:
Bacillus subtilis, Micrococcus lysodeikticus, Micro-
coccus pyogenes var albus and a strain of yeast.

814. Control of desoxyribonuclease activity.
Rosert N. FEINSTEIN AND FRANK O. GREEN.*
Argonne Natl. Lab., Lemont, Ill.

The assay of desoxyribonuclease (DNAse) ac-
tivity in tissues is complicated by the frequent
concomitant occurrence of a natural inhibitor
(DNAseIn) and a natural activator (DNAseA).
The DNAseA is heat-stable and so can easily be
measured, after heat-treating at 100°C. for 5 min.
to destroy all DNAse and DNAselIn activity, by
determining its effect on a pure DNAse source of
known activity. The activating effect is apparent
only if the viscosimetric or other short-time assay
method is employed. Both the DNAse and
DNAselIn of tissues are not only heat-labile but
also non-dialyzable. The existence of the inhibitor
can be demonstrated in several different ways:
a) If a carefully prepared (in the cold) extract of
blood cells, intestine, or a variety of other tissues
is added to an active DNAse source, the activity
is diminished. b) If such an extract is permitted
to ‘age’ under certain specified conditions, the

Volume 16

DNAse activity of the sample increases. c) If
such an extract is heated in a boiling water bath,
the extract will no longer inhibit an active source
but may actually activate it. A tissue preparation
which inhibits an active DNAse source may be
converted to an activating preparation by a)
heating; b) ‘aging;’ c) exposing to pH extremes; or
d) in certain cases, by simple dilution. (Work
performed under the auspices of the U. 8S. Atomic
Energy Commission.)

815. Insulin antagonist activity of serum in
diabetic acidosis. JAMES B. FrIe.p* anp
DeWitt Stetren, JR. Natl. Inst. of Arthritis
and Metabolic Diseases, Natl. Insts. of Health,
Bethesda, Md.

The cause of the insulin resistance associated
with diabetic acidosis has never been adequately
explained. Serum obtained from patients in di-
abetic acidosis has been found to contain insulin
antagonist activity measured by an in vitro tech-
nique. The method employed was the meas-
urement of extra glycogen accumulation in rat
hemidiaphragm after exposure to insulin (STap1IE
etal., Am. J. M. Sc., 218: 265, 1949). In most cases
studied this effect of insulin on the rat diaphragm
was abolished by preincubation with serum ob-
tained on admission from patients in diabetic
acidosis. From 10 to 72 hr. after institution of
therapy for diabetic coma, insulin antagonism
in serum could no longer be detected. The antago-
nist in non-dialyzable, and associated electro-
phoretically with the a- and 6-globulin fractions
of serum proteins. It does not interfere with the
binding of insulin-I'*! by rat diaphragm. Antago-
nism is readily demonstrable after incubation of
either insulin or diaphragm with active serum. It
was estimated that in the serum of one unusually
insulin-resistant subject, who received 21,000 units
of insulin in 24 hours, there was sufficient insulin
antagonist activity to abolish the effect of about
10,000 units of insulin.

816. Antibiotics as sources of anticar-
cinogens. JOHN B. Fietp, Doris A. FILuer,*
Louis T. Bascoy,* FRANcorsE CostTa* AND
ANGELA Boryczka.* Dept. of Medicine, Univ.
of Southern California School of Medicine and
Los Angeles County Hosp., Los Angeles.

An increasing number of culture filtrates from
various organisms are being found to have an in-
hibitory effect upon tumor growth. This labora-
tory has been studying filtrates and antibiotics
in both laboratory and clinical trials. Four agents
of greatest interest are crystalline antibiotics;
Actinomycin C obtained from Aspergillus
fumigatus, Fumagillin, also obtained from Asper-
gillus fumigatus, Azaserine obtained from a soil
Streptomyces characterized as o-diazoacetyl-L-

eo —=s = re FO ROOF UD lft ee CO

ume 16

c) If
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ay be
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March 1956

serine, and the fractions obtained from S.
Pastorianus. Given i.p. to mice with Sarcoma 180,
Actinomycin C in doses of 75-100 mg/kg/daily/7
days preduced consistent tumor inhibition so that
the average tumor diameter was oné-quarter to
one-half that of control tumors. In mice with RC
Carcinoma doses of 50-80 mg/kg/daily/7 days
gave a similar inhibition of tumor. In Leukemia
L 4946 there was some prolongation of survival
with 4-6 doses of 25-75 mg/kg. In the Harding-
Passey melanoma, 12 doses of 75 mg/kg produced
a tumor inhibition of 28%. In human trials in 35
individuals brief remissions have been induced
in only several cases of early Hodgkin’s disease,
one case of Hodgkin’s disease resistant to other
chemotherapy, and suggestive improvement was
noted in instances of multiple myeloma and acute
leukemia in children. Actinomycin D is currently
being evaluated and will be reported. Azaserine
is one of the most potent anti-tumor agents evalu-
ated in this laboratory. In mice with Sarcoma 180,
a dose of 32 mg/kg/day induced a tumor inhibition
of 91%; in mice with the RC Carcinoma it gave
an inhibition of 88%. Fumagillin given to mice
with Sarcoma 180 at a dose of 200 mg/kg/daily/7
days induced a tumor inhibition of 44%, and a
tumor inhibition of 39% with the RC Carcinoma.
The crystalline Actidione (6-(2-(3,5-dimethyl-2-
oxocyclohexyl)-2-hydroxyethyl) glutarimide gave
only borderline inhibition of Sarcoma 180. The
residual solids from S. Pastorianus are being
studied for tumor inhibition in Sarcoma 180 and
in the RC Carcinoma. All 4 antibiotics produced
a marked histological disruption of the tumors.
We have studied other mouse and rat tumors,
effect of delayed therapy, and combinations of
the antibiotics. (Supported by grants from the
Fndn. for Cancer Chemotherapy Research, the
Grace McCray and Cora Niles Memorial Funds.)

817. Isotopic and enzymatic studies of
thymine’ metabolites. Kay Fink, C.
McGavueauey,* R. B. HENDERSON* AND R. M.
Fink. VA Hosp., Long Beach, and Biophysics an
Physiological Chemistry Depts., Univ. of Cali-
fornia, Los Angeles.

After incubation of radiothymine with rat liver
slices, B-aminoisobutyric acid (BAIB) was de-
tected among the radioactive products (Federation
Proc. 14: 210, 1955). Further chromatographic
identifications now include the reductive inter-
mediates, dihydrothymine (DHT) and £-ureido-
isobutyric acid (BUIB); two oxidative products,
5-hydroxymethyluracil and_ uracil.5-carboxylic
acid; and more distantly related compounds, e.g.
alanine and glucose. In preliminary studies with
DHT-2-C™ as substrate, radioactive urea and
BUIB were identified, but whether labeled DHT
was converted to radiothymine remains equivocal.

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS 251

The particle-free supernatant from liver homo-
genates contains the enzymes for converting DHT
to BUIB and BUIB to BAIB (Federation Proc.
13: 207, 1954), and these activities were separated
by precipitating with 30% ethanol, extracting with
water, and adjusting to pu 5.7. The DHT-splitting
activity remained in solution, appeared to be
stable indefinitely at 5°, showed an apparent
optimum near pH 9, was effective with 6-methyl-
dihydrouracil, dihydrouracil, DHT, and dihydro-
orotic acid, and in the first three cases, the
preparation also catalyzed formation of dihydro-
pyrimidines from the corresponding ureido acids.
The pu 5.7 precipitate could hydrolyze BUIB and
B-ureidopropionic acid to BAIB and £-alanine,
respectively, but was ineffective with all other
ureido acids tested; it showed optimum activity
at about pH 6-7, and was relatively labile in aque-
ous solution. Addition of cyanate or carbamyl
phosphate could convert the @-amino acids to
ureido acids, but biological catalysis of the reac-
tion was not unequivocally demonstrated. (Sup-
ported in part by Cancer Research funds of Univ.
of California and by PHS Grant C-16669.)

818. Preparation and metabolic studies of
radioactive 5-hydroxymethyluracil. R. M.
Fink, R. E. Ciuine* anp Kay Fink. Depts. of
Physiological Chemistry and Biophysics, Univ.
of California, Los Angeles, and VA Hosp., Long
Beach.

Johnson and Litzinger were unsuccessful in
attempts to obtain 5-hydroxymethyluracil (HMU)
by condensation of uracil and formaldehyde
(J.A.C.S., 56: 1940, 1936), but the potential use-
fulness of such a reaction for isotopic studies
prompted the present reinvestigation. Ion ex-
change chromatography of uracil-formaldehyde-
HCl reaction mixtures yielded a crystalline
product which showed the elementary composition
of HMU. The product could be oxidized to uracil-
5-carboxylic acid or reduced to thymine and
dihydrothymine, and appeared to be identical
with eaminated 5-hydroxymethyleytosine in
chromatographic behavior, absorption maximum
and minimum, and stability in dilute acids.
Treating uridine or deoxyuridine with acidic
formaldehyde yielded products which ‘chromato-
graphed approximately as predicted for the corre-
sponding 5-hydroxymethyl nucleosides. In
preparing radioactive HMU, 1-2 mg. of uracil-
2-C™ and 20-30 ul. of 37% HCHO (acidified to
0.08 N with HCl) were heated in a sealed capillary
at 100° for 25 hr. and developed as a paper
chromatogram with H.O-sat’d. sec.- BuOH. The
HMU-2-C™ was eluted from the paper and in-
cubated with surviving rat liver, and two of the
radioactive metabolites have been identified
tentatively as 5-hydroxymethyluridine and uracil-

252 FEDERATION PROCEEDINGS

5-carboxylic acid. In similar metabolic studies
with the original uracil-2-C'4, uridine and urea
have been identified as radioactive products.
(Supported in part by Cancer Research funds of
Univ. of California and by PHS Grant C-1669.)

819. Transferase activity of 8-glucuronidase
preparations. WiLLIAM H. FisHMAN AND &.
GREEN. Cancer Research and Cancer Control Unit,
Tufts Univ. School of Medicine, Boston, Mass.
Experimental evidence is reported for the

catalysis of §-glucuronidase of the reaction;
RO-C.H50¢ + R’OH —_ ROH + R’0- CeH 0s.
The presence of certain alcohols in a B-
glucuronidase digest containing phenolphthalein
glucosiduronic acid produced, as compared to
controls, an increased liberation of phenol-
phthalein and a reduction in the freed glucuronic
acid. The molar difference between phenol-
phthalein and glucuronic acid agreed closely with
the value found for new glucosiduronic acid
formed. It was possible to determine the latter
since it remained in the aqueous phase after the
unhydrolyzed substrate had been extracted by
ethyl acetate. Both glucuronic acid liberated and
glucosiduronic acid formed were determined by
the method of Fishman and Green (J. Biol. Chem.,
215: 527, 1955). The extent of transfer under opti-
mum conditions was high; e.g., 0.191 of 0.214 um
of glucuronic acid released from the substrate, were
found in the new glucosiduronic acid. The reaction
was observed also at low ratios of acceptor to
donor. #-Glucuronidase prepared in varying
degrees of purity from liver, bacteria and snail
digestive juice possessed transferase activity.
The effects on both hydrolytic and transferase
activity of 8-glucuronidase of pH, time, tempera-
ture, inhibitor, substrate and enzyme concentra-
tion were similar. For these and other reasons, it
is believed that hydrolytic and transferase activi-
ties of 8-glucuronidase are properties of one
enzyme rotein.

820. Physico-chemical studies of p-tolyl azo
fibrinogen. JoHN E. FitTzGERALD AND WALTER
L. Kottun (introduced by ALFRED CHANUTIN).
Dept. of Biochemistry, School of Medicine, Univ.
of Virginia, Charlottesville.

The interactions and molecular stability of
fibrinogen coupled with nonpolar azo compounds
have been studied. Coupling fibrinogen (M =
330,000) with 0-25 p-tolyl azo groups per molecule
results in a progressive decrease in solubility.
Raising the pu or lowering the ionic strength
increases the solubility of the complex. In this
range the total clottability and the rate of clotting
is not affected. However, as the number of azo
groups per molecule are increased the clot becomes
more granular and less compactible. Ultracentri-

Volume 16

fuge analyses of the modified fibrinogen show the
presence of at least three components: a main
peak which has sedimentation characteristics
similar to fibrinogen; a smaller, faster moving peak
and rapidly sedimenting polymers. The patterns
also suggest the presence of a rapidly diffusing
small molecular weight component. Coupling of
more than 25 diazo groups per molecule of fibrino-
gen results in a marked decrease in solubility and
a corresponding loss in clottability. The ultra-
centrifuge patterns show that the main peak ig
smaller and broader and there is a corresponding
increase in the amount of rapidly sedimenting
material. The results obtained with less than 25
groups per molecule can be interpreted on the
basis of increased van der Waal’s attractive
forces. With more than 25 groups, however, there
is, in addition, a structural alteration of the
molecule.

821. Enzymatic conversion of propionate to
succinate. Martin Fuavin, H. Castro-
Menpoza* aNnpD Wixti1AM 8. Becx.* Dept. of
Biochemistry, New York Univ. College of Medi-
cine, New York City.

Evidence has been presented that propionate is
oxidized in animal tissues by a path involving
conversion to propionyl CoA and an ATP-
dependent carboxylation to methylmalonyl CoA,
followed by isomerization of methylmalonate to
succinate, and oxidation of succinate via the
citric acid cycle (Nature, 176: 823, 1955). ‘Chis
pathway has been demonstrated in all animal
tissues so far studied, but not in yeast autolysates,
extracts of Cl. kluyvert, or spinach. After 10-fold
purification, from pig heart extracts, of the en-
zyme system catalyzing the formation of methyl-
malonyl CoA from propionyl CoA, COs, and
ATP, propionyl CoA can no longer be replaced
by propionate and CoA, nor ATP by ADP, and
the presence of inorganic pyrophosphatase can
no longer be demonstrated. The requirement of
ATP for the synthesis of methylmalonyl CoA
from propionyl CoA and CO: indicates that phos-
phate bond energy is utilized in this reaction, but
the mechanism by which the energy is made avail-
able is not yet clear. The colorimetrically assayed
liberation of orthophosphate from ATP by this
purified fraction is the same, with or without
supplementation with crystalline pyrophos-
phatase, suggesting that formation of inorganic
pyrophosphate from ATP does not occur in the
carboxylation reaction. The orthophosphate
liberation depends completely on the addition of
both substrates, propionyl CoA and KHCO;.
No evidence has so far been found for a separate
enzymatic activation of CO2. A net formation of
succinate from methylmalonate, by rat liver ace-
tone powder extracts supplemented with ATP

ume 16

ow the
1 main
2ristics
ig peak
atterng
ffusing
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ibrino-
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ultra-
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of the

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in the
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Tr ace-

ATP

March 1956

and CoA, has been shown in experiments in which
succinate was determined by cytochrome C reduc-
tion in the presence of succinoxidase. Unlike
methylmalonate, chemically synthesized methyl-
malonyl CoA is converted to succinate without
addition of ATP, even after Dowex-1 or charcoal
treatment of the liver enzyme to remove protein-
bound nucleotides.

$22. Field methods for estimation of blood
cholinesterase activity. JosEPpH H. FLEISHER
aNnp C. StanLEY Woopson (introduced by D. B.
Ditt). Enzyme Chemistry Branch, Chemical
Corps Med. Labs., Army Chemical Center, Md.
The use of color changes of a pH indicator as an
approximate measure of blood cholinesterase
(ChE) activity has been suggested by Limperos
and Ranta (Science 117: 453, 1953); the procedure
was modified by Davies and Nicholls (unpublished
experiments). In further modification, activity
was related to the time necessary under specified
conditions to produce an amber color with brom-
thymol blue (pH 6.7). Reaction time has been
correlated with temperature of reaction (range:
15°-35°C) and degree of inhibition of ChE ac-
tivity, in the form of charts which permit evalua-
tion of ChE activity from fingertip blood samples.
Because of sex differences, different curves apply
to males and females. Without individual normal
controls, the lowest clearly distinguishable level
of whole blood ChE inhibition is 50%; if such
controls are available, lower levels may be dis-
tinguished. Analogous curves have been con-
structed with acetyl-8-methyl choline instead of
AcCh as substrate, which permit evaluation of
red cell ChE rather than total blood ChE inhibi-
tion in unseparated blood samples. Reagents for
the test can be prepared in dry form for use in the
field thus improving storage stability.

823. Triiodothyronine derivatives in urine
and bile. Eunice V. Fiock, JoHn H. GrinpLAy*
AND JESSE L. Botuman.* Depts. of Biochemistry
and Exptl.Surgery, Mayo Fndn., Rochester, Minn.
Radioactive triiodothyronine was administered

intravenously in doses of 50 uc/kg to dogs before

and after the establishment of a biliary fistula
with removal of the gall bladder, and again after
obstruction of the biliary fistula. Lugol’s solution
was given orally to each dog 1 or 2 days before
each injection of triiodothyronine. One dog ex-
creted 34.5% of the administered I'*! in the urine
within 24 hr. after the injection of triiodo-
thyronine. Four days after the biliary fistula was
made, and 42 days after the first injection, a second
dose of triiodothyronine was administered. The
dog excreted about the same amount of [!*! in
the urine as before, and in addition also excreted
24.0% of the I!*! in the bile. When a third injection

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

253

of triiodothyronine was given 10 days later, 2 hr.
after obstruction of the biliary fistula, larger
amounts were excreted in the urine. Fractionation
of the iodine compounds in the urine on a
kieselguhr column (Braascu, J. W., E. V. Fock
AND A. ALBERT, Endocrinology, 55: 768, 1954)
showed chiefly 4 peaks of radioactivity, corre-
sponding to triiodothyronine, an unidentified
compound, iodide, and the glucuronide of tri-
iodothyronine. The dog excreted 3.5, 6.5, 24.0
and 0.6% of the dose as these 4 constituents before
the biliary fistula was established and 3.9, 6.2,
23.4 and 3.7% afterwards. When the fistula was
obstructed more iodide and conjugate were ex-
creted. Dehepatized dogs, studied previously
excreted less iodide than normal dogs or dogs
with biliary fistula or biliary obstruction.

824. Glycolysis in the growing and spontane-
ously regressing Flexner-Jobling car-
cinoma. P. J. Fopor, P. ToMAsHEFsKY* AND
C. Funx. Dept. of Biochemistry, New York Med.
College and Funk Fndn. for Med. Research, New
York City.

There is no difference in the overall glycolytic
rates of regressing and nonregressing tumors in
the presence of glucose, HDP, pyruvate, DPN,
ATP, MgCle, phosphate and KF in concentrations
sufficient to completely inhibit ATPase. However,
if KF is omitted, glycolysis, measured by the
liberation of COz from bicarbonate-phosphate
buffer, starts with a high initial rate and drops
to zero after 20-25 min. Increased amounts of
HDP, pyruvate and DPN fail to restore activity
and are without effect on the rate of the reaction.
In nonregressing tumors glycolysis continues for
between 60-120 min. The curve of CO: liberation
by regressing tumors is identical with that pro-
duced in the presence of ATP, MgCle, bicarbonate
and phosphate only. Direct determinations of
phosphate liberated by enzymatic action showed
that ATPase in regressing tumors is considerably
higher than in growing tumors. Higher than
normal ATPase activity was also found in tumors
which, after reaching maximal size, start to dis-
integrate from within. In the latter glycolysis is
lower than in the still growing tumors but not as
low as in the regressing ones. Thus, increased
ATPase prevents in varying degrees the utiliza-
tion of glucose by tumors which either regress or
disintegrate in situ. The possible role of other
dephosphorylating enzymes likely to act on
glycolysis intermediates is under investigation.
All determinations pertain to the non-necrotic
outer tissue layer of the tumors.

825. Physical particle per plaque ratios ob-
served for human poliomyelitis viruses.
J@RxcEN FocH anp CaritTon E. ScHWweRDT

254

(introduced by WeNpELL M. Stantey). Virus

Lab., Univ. of California, Berkeley.

Quantitative measurements of physical particle
concentration by analytical electron microscopy
and of plaque forming unit (PFU) concentration
by tissue cultue plaque assay have made possible
estimates of physical particle per plaque ratios
for strains of type 1, 2 and 3 poliomyelitis virus.
Virus has been harvested from monolayer cultures
of monkey kidney epithelial cells and human
amnion cells, transferred to and concentrated
in a volatile salt solution, assayed in amnion and
monkey kidney monolayer cultures for PFU and
sprayed for electron microscope counting. Under
these conditions ratios ranging from 30 to 60
countable particles per plaque have been obtained.
This is in contrast to the 1000+-500 ratios obtained
earlier with virus concentrates purified from
infective tissue culture fluid. Successful reduction
of this ratio to its present lower value appears to
depend, in large part, upon the conditions of
virus propagation, harvest and assay, the method
of virus concentration and partial purification
for quantitative electron microscopy, and the
plating efficiency of the tissue culture system used
for assay. Experiments are being done to see
whether or not it is possible to reduce these lower
ratios still further by changes in experimetal
conditions and by consideration of the thermal
inactivation of the virus during its propagation in
tissue culture at 37°C.

826. Brain strandin. Jorp1 Foucu, J. A. MEATH
AND 8S. Bococu. McLean Hosp. Research Labs.,
and Harvard Med. School Waverley, Mass.
Strandin (J. Biol. Chem., 191: 819, 1951) has been

prepared as a substance homogeneous by electro-

phoresis and ultracentrifugation. N: 2.9% N.E.:

1150. Conditions have been found for its stepwise

hydrolysis by a procedure in which different con-

stituents are released gradually, leaving the rest
of the strandin molecule as an undialyzable ‘core.’

Thus, at any point, the former can be separated by

dialysis. An aqueous solution of strandin as free

acid is heated at 100°. After 20 m. about 3 of the
chromogenic group (J) has been split off. After
recrystallization, J consists of needles. Its proper-
ties, composition, color-producing reactions and

x-ray diffraction picture (kindly run by Prof. G.

Blix), show it to be akin to or identical with,

ovine sialic acid. J is combined to the rest of the

strandin molecule by a reducing group. The treat-
ment of the undialyzable core with 0.09 HCl at
100° for various lengths of time, followed by
treatment with more concentrated acid solutions,
results in the gradual release of the constituent
sugars. These have been identified chromato-
graphically as being exclusively galactosamine,
galactose and glucose. They are released in the

FEDERATION PROCEEDINGS

Volume 1§

stated sequence, with considerable overlapping,
both as free sugars, and as different oligosac-
charides. Finally the core is reduced to fatty
acid(s) and ‘sphingosine,’ with some suggestion
of the possible presence of an unidentified other
constituent. (Aided by Grant BCH-11 et seq,
from the American Cancer Society).

£27. Action of protamine on cellular electro.
lyte transport. E. C. Fou.kes* np

BENJAMIN F. Mruuer. May Inst., Jewish Hosp.,

and Depts. of Physiology and Medicine, Univ,

of Cincinnati, Cincinnati, Ohio.

In extending experiments on ion permeability
of yeast (FouLKES, J. Gen. Physiol.) separation of
anion and cation movements was attempted.
Salts of high molecular weight ions were used for
this purpose. We have now observed that, unlike
in yeast, K transport in rabbit kidney, rat di-
aphragm and frog sartorius is inhibited by
protamine, a large cation. Tissues were depleted
of K in cold saline. Net K uptake and Na loss were
then measured in Ringer at 22° or 37°C in Qs,
Addition of 4th volume isotonic protamine salt
solution inhibits K uptake without affecting Na
extrusion or respiration. Smaller concentrations
of protamine produce lesser inhibition of K trans-
port. The fall of intracellular Nat is counter-
balanced approximately by uptake of protamine
salts. Hydration of tissues is little affected.
Protamine uptake occurs at 0°C and is therefore
independent of active movement of other cations.
Washing of the tissue will not reverse the inhibi-
tion after exposure to protamine. Polylysine and
certain basic dyes appear to behave like
protamine. Other basic compounds and acidic dyes
are inactive. Protamine also inhibits p-amino-
hippurate and Diodrast accumulation by kidney
slices; the system shows the same sensitivity as K
transport towards protamine. Separation of
movement of Na and K against concentration
gradients points to independent transport mecha-
nisms for these ions in the tissues studied. The
results further confirm the close relationship
between K transport and the accumulation of
organic anions (pAH, etc.) by kidney slices.

828. Synthesis of isomeric thymine pento-
furanosides, including ‘spongothymidine.’
Jack J. Fox anp NaisHuN CHana YuNG.*
Sloan-Kettering Inst. for Cancer Research, New
York City.

In a previous report (Federation Proc. 13: 702,
1954) we described the use of a mercuri-thymine
derivative for direct N'-glycosidation to thymine
nucleosides. Improvement of this procedure has
led to the relatively facile synthesis of 1-D-ribo-
furanosylthymine (I) and _ 1-D-xylofuranosyl-
thymine (II). Both these nucleosides give the same

TE aS el ll le a ee a ae er ee

— i . |

ume 1§

ping,
igosac-
» fatty
gestion
1 other
et seq.

lectro-«
"AND
Hosp.,
Univ,

ability
tion of
mpted.
sed for
unlike
rat di-
ed by
apleted
Ss were
in On.
ne salt
ing Na
rations
trans-
unter-
tamine
fected.
erefore
ations.
inhibi-
ne and
e like
ic dyes
amino-
kidney
yy as K
on of
tration
mecha-
d. The
onship
‘ion of
PS.

pento-
dine.’
YUNG.*
r, New

3: 702,
1ymine
1ymine
ire has
)-ribo-
unosyl-
e same

March 1956

dialdehyde upon oxidation with metaperiodate. The
former, (I), is identical with a ribosylthymine

prepared enzymatically by Lampen. Determina-,

tion of the configuration at the glycosidic center
of I was accomplished by epimerization of the
2’ hydroxyl group via a cyclonucleoside inter-
mediate (MICHELSON AND Topp, J. Chem. Soc.
816, 1955). This inversion can occur only if I is of
the beta configuration. Thus, the 5’-trityl deriva-
tive of I was prepared and treated with
methanesulphonyl] chloride to give a syrup (III)
(probably a mixture of 2’ and/or 3’ mesyl nucleo-
sides). Treatment of III with methanolic am-
monia followed by acid hydrolysis gave a crystal-
line nucleoside (IV) identical in all respects with
‘spongothymidine’ previously isolated by Berg-
mann and Feeney (J. Org. Chem. 16: 981, 1951)
from certain Carribean sponges. This reaction
proves conclusively that I, II, and IV are beta
nucleosides, I being a true 5-methyluridine.
Further, by virtue of the non-identity of IV with
I or II and since a lyxofuranosylthymine cannot
possibly arise from the above-mentioned epi-
merization reaction of I, the identity of
spongothymidine (IV) must be 1-8-D-arabino-
furanosylthymine. This is in accord with the
recent independent findings of Bergmann and
Burke (J. Org. Chem. 20: 1501, 1955).

829. Isolation from rat skin of a possible in-
termediate in cholesterol synthesis. Ivan
D. Frantz, Jr., ANN G. Davipson* AND ELINOR
Duuit.* Cardiovascular Research Lab., Dept.
of Medicine, Med. School, Univ. of Minnesota,
Minneapolis, Minn.

By a previously described method (Circulation
12: 501, 1955) we have isolated from 14 kg of rat
skin 500 mg of a sterol which we have not yet
been able to identify with a known compound.
This sterol is chromatographically separable from
cholesterol, cholestanol, A’-dehydrocholesterol,
lanosterol, A’-cholestenol (‘lathosterol’), A8@4-
cholestenol, and zymosterol. It is less polar than
cholesterol, but more polar than lanosterol. It is
precipitable with digitonin. When treated with the
Liebermann-Burchard reagent it produces an
immediate purple color. The maximum intensity
of absorption is reached at 14 min., and is ap-
proximately four times that given by an equal
weight of cholesterol at its maximum. At 30 min.
the color has faded to about 3 of the maximum,
in contrast to a fading to 4 in the case of
lathosterol. The melting point is 140°-141°, and
the rotation in chloroform +28°. By perbenzoic
acid titration the compound appears to have two
double bonds, but no conjugated double bond sys-
tem is detectable by ultraviolet absorption. The
infrared absorption differs in minor details from
the patterns of cholesterol and lathosterol. We

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

255

have been unable to separate the compound
chromatographically from one of the ‘higher
counting companions’ of cholesterol obtained
from liver slices incubated with labeled acetate.
Cocrystallization also fails to provide evidence of
inhomogeneity. This higher counting companion
is readily convertible to cholesterol by liver slices.

830. Production of bile acids from cholesterol
by liver mitochondria. DoNALD S. FREDRICK-
son (introduced by Harry D. BarRnsTEIN).
Natl. Insts. of Health, Bethesda, Md.

Several bile acids as well as neutral products
have been isolated and partially identified follow-
ing incubation of cholesterol-C" in a system con-
taining mouse liver mitochondria previously
shown capable of the production of C'O. from
cholesterol-26-C'* (Federation Proc. 13: 212, 1954).
In the saponifiable material recovered following
a typical 3-hour incubation of cholesterol-4-C",
6% of the original substrate radioactivity was
present in a heptane-soluble fraction (I), and 3%
in an ether-soluble fraction (II). Essentially all
of this material was alkali-extractable and pre-
cipitable as the Doubilet salt. Fraction II was
further separated into 3 components by paper
chromatography. One of these was identical in
polarity to cholic acid and a second to desoxy-
cholic or chenodesoxycholic acid. However,
recrystallization experiments indicated that
neither radioactive component was identical to
these known compounds. The third, a more polar
acid, was digitonin-precipitable. Fraction I radio-
activity was further resolved by counter-current
distribution into several components having
partition co-efficients similar to bile acids. The
bulk of this radioactivity was Girard-T reactive.
The sidechain length of at least part of the ring-
labeled acid products may be assumed to be
greater than 24 carbons since acids containing a
terminal radioactive carbon have been recovered,
in a lower percentage yield, following incubation
of cholesterol-26-C'*. An enzymatically-produced
neutral compound accumulating during the reac-
tion has been tentatively identified as 25- or 26-
hydroxycholesterol. Up to 25% of the cholesterol
appears to be esterified during the incubations.

831. Tissue heparin and mast cells in rats
and rabbits. L. Freeman,* L. Marx* anp
W. Marx. Depts. of Biochemistry and Nutrition,
Univ. of Southern California School of Medicine,
Los Angeles.

Tissue heparin contents were measured in vari-
ous organs of adult rats and rabbits, using a
method adapted to relatively small tissue samples
(1-5 gm fresh tissue) and to relatively low heparin
concentrations (as low as 0.5-1.0 USP unit/gm).
Mast cell counts were made on the same organs.

256

We found that, among the rat tissues examined,
kidney and thymus had the highest, and liver
the lowest heparin contents; lung, intestine and
spleen showed intermediate values. The heparin
concentrations of rabbit organs were lower than
those of the corresponding rat tissues. Only in
some instances did mast cell numbers follow a
similar trend; in part, a different pattern was
observed, when the various organs were compared.

832. Inhibition by dinitrophenol of the phos-
phate-dependent catalysis of sulfite oxida-
tion by metals. Irwin FRIDovicH* AND PHILIP
Hanpier. Dept. of Biochemistry, Duke Univ.,
Durham, N. C.

The aerobic oxidation of sulfite by cyanide-
treated xanthine oxidase, at pH 7, proceeds vigor-
ously in phosphate but very slowlyin TRIS buffer.
The reaction appears unrelated to the flavin or
protein components since ashed enzyme serves
equally well. Indeed, 4.5 X 10-* M MoO; in 5 X
10-* M cyanide, as well as 4.5 X 10-* M Fe**,
also catalyze rapid aerobic oxidation of SO;7 in
phosphate but not TRIS buffer. Whereas molyb-
denum catalysis is effective only in the presence
of cyanide, Fet* catalysis is reduced only 50%
by omitting cyanide. Neither metal is effective,
nor is cyanide treated xanthine oxidase, at a
pH >7.6. With either metal as catalyst in 0.11 M
phosphate, 10-? M DNP almost completely in-
hibits sulfite oxidation as does much smaller
concentrations of throxine. Relatively low con-
centrations of pyrophosphate and dithionate,
but not of sulfate, arsenate or chloride, replace
orthophosphate in these systems and it appears
that the slow autocatalytic rate curves observed
in TRIS and in DNP-inhibited systems may be
due to the generation of dithionate. All experi-
ments were conducted in the presence of 0.01%
versene Fe-III. It is tempting to speculate that
these studies afford a model for oxidative phos-
phorylation as observed in mitochondrial prepa-
rations. Reoxidation of the reduced metal-phos-
phate complex may result in an _ energetic
phosphate which might be transferred in the
presence of suitable acceptor systems. Dinitro-
phenolate may be presumed to displace phosphate
as a complex with the reduced metal. Studies
designed to test these possibilities are in progress.

833. Enzymes and amphibian metamorphosis.
Eart FRIEDEN (introduced by Donatp H.
Coox). Dept. of Chemistry, Florida State Univ.,
Tallahassee.

A survey of possible enzymatic changes during
normal and induced metamorphosis of amphibia
is in progress. Extensive studies of changes in
arginase activity have been recently reported
(Dorin, J. L. anp E. Frrepen, J. Biol. Chem.,

FEDERATION PROCEEDINGS

Volume 15

Dec. 1955). Further studies of other important
enzyme systems have been made. Fortified homo.
genates of tadpole (Rana Hechsheriz) liver, tail,
and heart oxidize succinate readily. However, no
significant differences in succinate oxidation per
unit of fresh tissue was observed during normal
or induced metamorphosis. Other substrates
such as glutamate, pyruvate, maleate, acetate,
aspartate, $-hydroxybutyrate and others are
only sluggishly oxidized by either tail or liver
preparations, even when fortified with coenzyme
concentrates. Results with other enzymes will
be presented. The data suggest that there may be
little significant change in this group of enzymes
during normal and induced amphibian meta-
morphosis. (This research was supported by a
Public Health Service grant.)

834. Effect of testosterone propionate upon
the incorporation of glycine into mouse
kidney protein. Epwarp H. FrriEepEn. Arthritis
Research Lab. and Cancer Research and Cancer
Control Unit, Tufts Univ. School of Medicine,
Boston, Mass.

The effect of testosterone propionate (T.P.)
upon the incorporation of labeled glycine into
kidney protein in vitro has been studied in several
strains of mice. Kidney slices from treated and
untreated animals were incubated at 38.7° in
Krebs-Ringer phosphate 1.0 mm in glycine-1-C¥,
Incorporation into kidney protein of both groups
was found to be linear for 3 hr.; a 2-hr. incubation
time was generally used. For female A Cloudman
mice, the maximum response to T.P. occurs about
24 hr. after the second of 2 daily injections (1.0
mg T.P. in 0.1 ml sesame oil). Under these condi-
tions the rate for treated animals averaged 70%
higher than that of control groups. The effect of
T.P. appears to be independent of the amount
injected over the range 0.1-5.0 mg/day. The addi-
tion to the medium of an equal volume of serum
from untreated mice virtually abolishes the differ-
ence between control and T.P.-injected groups.
When the glycine content of the medium was
varied, the effect of T.P. was found to be sub-
stantially unchanged below 1.0 mm. Above this
concentration, however, its effect diminishes, s0
that the difference between control and treated
groups disappears around 5.0 mm glycine. These
observations suggest that the effect of the steroid
is to facilitate the accumulation of glycine into
the cell from a relatively glycine-deficient me-
dium. (Aided by research grants from the Natl.
Inst. of Arthritis and Metabolic Diseases, Natl.
Insts. of Health.)

835. Isolation and physiological action of
(+) carnitine. S. FrrepmMan, A. B. GALun
AND G. FRAENKEL (introduced by W. C. Rose).
Dept. of Entomology, Univ. of Illinois, Urbana.

lume 1§

portant
| homo-
ar, tail,
ver, no
ion per
normal
strates
acetate,
Ts are
or liver
enzyme
es will
may be
nzymes

meta-
d by a

> upon
mouse
l rthritis
Cancer
edicine,

(T.Pa
ne into
several
ed and
8.7° in
e-1-C¥,
groups
ubation
yudman
s about
ms (1.0
» condi-
ed 70%
ffect of
amount
e addi-
f serum
> differ-
groups.
im was
be sub-
ve this
hes, 80
treated
. These
steroid
ne into
nt me-
e Natl.
, Natl.

ion of
GALUN
Ross).
bana.

March 1956

(—) Carnitine, a quaternary ammonium com-
pound having the formula (CH;); NtCH:-
CHOHCH.COO—, is found almost universally
in animal tissue, and is known to be a requirement
for growth of the larva of the bettle, Tenebrio
molitor L. Its function in the metabolism of this
insect and of animals generally is still almost
completely unknown. Since the naturally oc-
curring compound is levo-rotatory and dl-
carnitine (which is readily synthesized) appears
to be easily utilized, it is of interest to know
whether the dextro-rotatory form has similar
activity when tested on 7’. molitor. A microor-
ganism having the ability to use carnitine as the
sole carbon source was selectively isolated from
a soil sample by the enrichment technique, and
was grown on a medium containing dl-carnitine
plus inorganic salts. It was found that after 24-48
hr. of growth ina highly aerated culture flask the
medium became optically active. (+) Carnitine
was isolated from this medium, and the purified
compound was found to have an [a]> = +22.3.
Derivatives of this compound had melting points
similar to those of the (—) compound. The activity
of this compound when tested as a growth factor
for T. molitor was completely negative. Tests
made on (—) carnitine showed it active at levels
of 0.75 ug/gm of diet, while (+) carnitine was
inactive at levels up to 50 ug/gm of diet.

836. Isolation and characterization of two
new urinary steroids. Davin K. FukusHm™a,
Evetyn D. Meyer,* Eruen, ASHWORTH* AND
T. F. Gatiacuer. Sloan-Kettering Inst. for
Cancer Research, New York City.

Two new steroids with the 17a,20a-glycol side
chain have been isolated from human urine. The
compounds were characterized by infrared spec-
trometry and mixture melting point determination
with authentic samples prepared by partial
synthesis. Pregnane-3a,118,17a,20e-tetrol, m.p.
134-138°; [a]? +10.2° (chloroform) ; 3,20-diacetate,
m.p. 214-219°; [a]® +16.2° (chloroform), was
isolated from the urine of a man with adrenal
hyperplasia and a man with Cushings’ syndrome.
11-Ketopregnane-3a,17a,20a-triol, previously re-
ported by Swiss investigators, was also isolated
and characterized from the same urines. Allopreg-
nane-3e,17a,20a-triol, m.p. 226.5-229.5°, [a]?!
—13.2° (chloroform); 3,20-diacetate, mp.
126-128°, [a] —15.7° (chloroform), was isolated
from the urine of a man with adrenal hyperplasia
and a woman with adrenal virilism. The allopreg-
nanetriol was isolated from the same urine from
which 3a,17a-dihydroxyallopregnane-20-one was
obtained. These two compounds represent the
only allopregnane derivative with a 17a-hydroxyl
group thus far isolated from human urine.

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

257

837. Mammalian transamidinase. Maria
Fup. Dept. of Biochemistry and Nutrition,
Graduate School of Public Health, Univ. of Pitts-
burgh, Pittsburgh, Pa.

Transamidinase from hog kidney (Federation
Proc. 13: 215, 1954) was purified approximately
200-fold by repeated acetone fractionations at
pH 7 and 5.5, ammonium sulfate fractionation at
pH 6.3, and adsorption on calcium phosphate gel.
Nucleic acids were eliminated by surface pre-
cipitation at pH 4.6-5.0 on acetate-charged
Amberlite IR-45 or Amberlite IR-4B (Communi-
cation, Third International Congress of Bio-
chemistry, p. 36, 1955). The mechanism of action
of transamidinase was investigated with these
preparations. After exhaustive dialysis, incuba-
tion of non-labelled arginine and ornithine-2-C-14
at pH 7.2 in the absence of an amidine acceptor
led to the formation of arginine containing C-14.
This exchange suggests two possible mechanisms
of amidine transfer: a) An amidino-enzyme com-
plex is formed which then reacts with a suitable
amidine acceptor, or b) A substituted amidino-
enzyme complex, i.e. R’-amidino-enzyme, is
formed. R’ is subsequently replaced by R” leading
to R”-amidine plus R’, plus enzyme. Such inter-
mediates are probably involved in the transfer
reactions from arginine to glycine, from glyco-
cyamine to ornithine, and from arginine to
ornithine. R’ and R” represent the respective
amino acids. In this event the reaction may have
wider significance, and there may exist other
amidine acceptors and donors. Evidence was
obtained that lysine can act as an acceptor leading
to the formation of homoarginine. Serine and
B-alanine are not acceptors which suggests that
the acceptor must contain both an w- and an a-
amino group. Work is in progress to establish
whether 2,4-diaminobutyric acid and 2,3-diamino-
propionic acid can act as acceptors. An interesting
possibility exists that water may also function
as an amidine acceptor leading to an arginase-like
activity since all preparations, so far, exhibit
arginase activity at pH 8.8 in the presence of
Mn**, (Supported by a grant from the Public
Health Service.)

838. Alloxan-adducts in pre-diabetic states,
C. Funk, P. Tomasnerskxy,* T. T. Inryz,*
AND R. Asoopy.* Funk Fndn., New York City.
Some years ago, the presence of blood sugar re-

ducing substances, possibly guanidine derivatives,

demonstrated in yeast and other sources (C. Funk

AND H. B. Corsirt, Proc. Soc. Exper. Biol. &

Med. 20: 422, 1923; J. C. Drummonp anp C.

Funk, Biochem. J.8: 598, 1914). In this preliminary

report we have detected alloxan-like substances

in urine, spleen, and pancreas. We suggest, there-
fore, that the presence of these antagonistic

258

substances, i.e. alloxan and guanidine derivatives,
in foods, or their formation by enzyme action,
may be significant in the etiology of early diabetes.
This pertinent ensyme pattern seems to vary a
great deal from one subject to another. In accord-
ance with the known data on the very rapid
disappearance from the blood of injected alloxan,
it is perhaps, significant that this substance com-
bines almost instantly, with tissue constituents
such as cystine, cysteine and glutathione. We
have, at present, prepared eight such adducts.
They are now being purified and tested biologi-
cally. The somewhat unpredictable behavior of
alloxan in individual animals may be explained
by the variation in the kind and rate at which the
natural adducts are formed. Thus, the alloxan-
cysteine adduct is much less diabetogenic than
the equivalent amount of alloxan. We have iso-
lated from normal urine and spleen substances,
apparently alloxan adducts, which are now being
further investigated. Our preliminary data suggest
that crude insulin and glucagon may also contain
alloxan-like substances.

839. Influence of methyl testosterone and of
dl-ethionine on canine liver lipids and
serum lipids and lipoproteins. Rogpert H.
Furman, Leonarp N. Norcia, CHar.es W.
Rosinson AND I. ERNEstT GONZALEZ, (intro-
duced by Max N. Hurrman). Oklahoma Med.
Research Fndn., Oklahoma City.

When dogs are fed, for periods of 10 days or
more, either methyl testosterone, 200 mg, or pL-
ethionine, 250-500 mg, 6 days per week, similar
and marked reductions in serum lipid and lipo-
protein concentrations occur. A total of 6 courses
of methyl] testosterone were administered to 2 male
and 2 female mongrel dogs weighing 14-21 kg.
The percentage fall of serum lipids from control
values was as follows: total cholesterol, 72-50%;
lipid phosphorus, 63-52%; ‘high density’ lipo-
protein (flotation rate characteristic —S 1-10 in
solvent of density 1.21), 64-51%. ‘Low density’
lipoprotein (—S 20-40) changes were always in
the same direction but there was considerable
variability. The serum lipid changes resulting
from pt-ethionine administration were similar
and are in agreement with those previously pub-
lished by Chaikoff’s group (Science, 120: 317, 1954).
When serial serum lipid determinations indicated
maximal depression of serum cholesterol,
laparotomy with liver biopsy was carried out.
Biopsy was repeated when serum lipids had
reached control or maximal values after medica-
tion was discontinued. Both methyl testosterone
and pt-ethionine administration led to virtual
depletion of liver lipid phosphorus (less than 5%
of normal). The range of values for both groups
in 10 biopsies during treatment of 7 animals was

FEDERATION PROCEEDINGS

Volume 16

from 0.04 to <0.01 mg/gm wet weight of liver.
Similar but less marked reduction in liver total
lipid and total cholesterol content was also noted,
(Supported by Public Health Service Grants
H-1429 and H-1694.)

840. Enzymatic inactivation of folic acid.
SipnEyY FuTrerRMAN (introduced by Joun C,
Keresztssy). Natl. Insts. of Health, Bethesda, Md.
When folic acid is incubated with liver extracts

the loss in Streptococcus faecalis R activity ig

accompanied by the appearance of stoichiometric
amounts of an acetylatable aromatic amine. The
spectral and chromatographic properties of the
amine are identical to those of p-aminobenzoyl-
glutamic acid. Enzymatic activity of chicken liver
extracts is lost upon dialysis and restored by the
addition of boiled juice or by a combination of the
following supplements: DPN (SILVERMAN,

KERESZTESY AND Kovat, J. Biol. Chem. 211: 58,

1954), ATP, Mn**, and citrate. The supplemented

system containing 2007 of folic acid in a final

volume of 1.5 ml inactivates approximately half
of the substrate in 1 hr. Aminopterin is not in-
activated by the extract and strongy inhibits the
inactivation of folic acid, 0.25y of aminopterin

causing 90% inhibition in the presence of 200,

of folie acid.

841. Phosphorylase and Q-enzyme in develop-
ing corn kernels. Hipetsucu Fuwa (intro-
duced by A. K. Batts). Dept. of Biochemistry,
Purdue Univ., Lafayette, Ind.

As a step to some insight into the formation
of starch in corn endosperm, and to investigate
some of the possible factors controlling the ratio
of amylose to amylopectin in starch, changes
in the phosphorylase and Q-enzyme activities
were followed during the period when starches are
being formed in the kernels of starchy and waxy
varieties of corn. Kernels homozygous for Wx and
wx were collected from single crosses, respectively.
The essential genetic difference between these
starchy and waxy single crosses is the single gene
determining the waxy condition of the endosperm.
There was no marked difference between the
starchy and waxy varieties in respect to the ratio
of phosphorylase and Q-enzyme activities
throughout the period tested (12-25 days after
pollination). Yet the waxy variety produced no
permanent amylose whatever, while the starchy
variety laid down a considerable fraction (ca.
20%) thereof. Thus the ratio of phosphorylase
and Q-enzyme does not determine the ratio of
amylose to amylopectin. Obviously, other factors
enter into the picture, and it seems probable that
they act in addition to the system of phos-
phorylase and Q-enzyme rather than as a separate
synthetic apparatus. (The assistance of Dr. A. J.

ume 16

liver,
- total
noted,
srants

acid,
HN C,
la, Md.
‘tracts
‘ity is
metri¢
e. The
of the
nzoyl-
n liver
by the
of the
RMAN,
L1: 53,
1ented
. final
y half
ot in-
its the
pterin

F 2007

elop-
intro-
nistry,

.ation
tigate
ratio
anges
vities
es are
waxy
x and
ively,
these
| gene
perm.
. the
ratio
vities
after
d no
urchy
(ca.
ylase
io of
ctors
that
phos-
arate
A. J.

March 1956

Ulistrup of Purdue Univ. is most gratefully
acknowledged. The work was aided by a grant
from the Corn Industries Research Fndn.)

$42. Metabolism of N" labeled glycine during
administration of growth hormone. O. H.
GAEBLER AND RacHev LineR.* Edsel B. Ford
Inst. for Med. Research, Detroit, Mich.
Experiments with N' labeled glycine were
carried out during nitrogen balance studies, with
and without growth hormone, in a normal dog and
in a depancreatized one receiving a constant dose
of insulin. In the normal dog the percentage of
ingested N!* excreted during the first 48 hr. was
comparable with that previously observed in this
laboratory (J. Biol. Chem. 196: 1, 1952), although
the present dose of labeled glycine (20 mg/kg;
31.5 atoms % excess N!, ingested with the single
daily feeding) was twice as great. Increasing the
dose of labeled glycine provided a more sensitive
method of detecting the profound effect of growth
hormone on output of ingested N'* without other-
wise modifying the result. In the depancreatized
dog, the dose of labeled glycine was doubled
again, by giving the above amount with each of
the 2 daily feedings. Even so, effects of growth
hormone (2.5 mg daily) on N'* output as well as
on total nitrogen excretion were small and
transient. The combined effect of growth hormone
plus an increase in the daily dose of insulin was
also disappointingly small. N'5 incorporation into
fibrinogen was only slightly increased by growth
hormone, both in the normal and in the depan-
creatized dog.

843. Adaption of Aerobacter to rhamnose.
R. L. Garner AND Grace I. Finx.* Dept. of
Biological Chemistry, Univ. of Michigan Med.
School, Ann Arbor.

Cultures of Aerobacter aerogenes were grown in
a glucose:peptone:yeast extract media and the
cells thus obtained were washed 3 times with 0.9%
NaCl solution. Suspensions of these cells in a
0.05 m phosphate buffer of pH 7 ferment glucose
readily, but are refractory toward rhamnose for a
period of approximately 1 hr. After 1 hr., a de-
struction of rhamnose is initiated and proceeds
with an increasing rate until the substrate is
exhausted. Cells adapted to the fermentation of
rhamnose were obtained by incubation of aqueous
suspensions of washed cells with rhamnose at
37°C. for 1 hr. after which they were collected
and again washed thoroughly with saline. During
the adaption period, there was no apparent cell
proliferation as measured by the determination
of the optical density of serial dilutions of the
preparations. The adapted cells exhibited an
immediate and rapid utilization of rhamnose
which could be determined by sugar destruction

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

259

or by oxygen consumption. The adapted cells
likewise exhibited an ability to metabolize fucose,
a property which was not detectable in the un-
adapted preparations. The substrate specificity
of the enzymes present in extracts of the adapted
cells and the role of phosphorylation in methyl-
pentose metabolism will be diseussed.

844. Adrenal cortical reserve and cortical
metabolism in normal and toxemic preg-
nancy. JOSEPHINE B. Garst, Nicuotas S.
AssaLi* AND MarGaret R. Henuey.* Dept. of
Obstetrics and Gynecology, Med. Center, Univ.
of California, Los Angeles.

Previous studies have shown that the plasma
levels of 17-hydroxycorticosteroids are higher in
normal and toxemic pregnancies than in non-
pregnant individuals. In an attempt to explain
these elevated levels, a study was made of adrenal
cortical reserve and cortisol metabolism. Forty
mg of ACTH were given intravenously over 6-10
hr., plasma 17-hydroxycorticosteroids being de-
termined every 2 hr. In general the values for 12
normal pregnant patients showed a nearly linear
rise during the time studied (possibly not maximal
at 10 hr.); the values for 12 toxemic patients
showed somewhat less marked increases with
maxima in 6 hr. or less. These data show that the
adrenal cortex in normal pregnancy is not stimu-
lated maximally. They also suggest that the
responsiveness of the adrenal cortex in toxemia of
pregnancy is reduced, or else that the production
of this type of corticoid is impaired. Seventy-five
mg of cortisol were given intravenously over 4 hr.
to 8 normal and 5 toxemic pregnant patients.
Blood was drawn at 0, 1, 2, 4, 6 and 8 hr., and
plasma 17-hydroxycorticosteroids determined. At
1 hr. an average of only 6.3% of the exogenous
cortisol remained in the plasma of the normal
patients and 5.6% in the plasma of the toxemic
patients; at 8 hr. the values approached those
found at zero time. It would appear that the
ability of the body to handle large amounts of
cortisol is not impaired in either type of patient.

845. Reconstructed systems of glycolysis and
oxidative phosphorylation. S. Garr,* I.
Krimsky* anp E. Racker. Div. of Nutrition
and Physiology, Public Health Research Inst.,
New York City.

In attempts to elucidate the inhibition of fer-
mentation by respiration (Pasteur effect) and the
inhibition of respiration by fermentation in tumor
cells (Crabtree effect), a reconstructed system
consisting of glycolytic enzymes and actively
respiring liver mitochondria was studied. It was
found, as shown previously by other investigators,
that the oxidation of glutamate by mitochondria
is stimulated in the presence of a phosphate

260

acceptor system such as glucose, hexokinase and
catalytic amounts of ADP. At low ADP con-
centrations (3 X 10-‘ m), the addition of glucose
and the complete glycolytic system resulted in a
pronounced inhibition of mitochondrial res-
piration. There was no inhibition of respira-
tion at high ADP concentrations (2 X 107? m)
or when glycolysis was prevented by omission
of a single glycolytic enzyme, e.g. phosphofruc-
tokinase. The inhibition was partly or com-
pletely released by 2,4-dinitrophenol. These
findings are in line with a hypothesis that limiting
amounts of ADP are shared by the intra- and
extra-mitochondrial systems, and shuttle back
and forth between them. Thus, an active gly-
colytic system may deprive respiration of essential
ADP and vice versa. In the presence of glycolytic
enzymes and actively respiring mitochondria,
pyruvate can serve as a phosphate acceptor.
Inorganic phosphate is taken up and phospho-
enolpyruvate and other phosphate esters
accumulate. These reactions represent a direct
reversal of the glycolytic steps involved. Forma-
tion of phosphoenolpyruvate from ATP and
pyruvate occurs readily when the ATP/ADP ratio
is high, as is the case during oxidative phos-
phorylation. Subsequent steps in the reversal of
glycolysis favor additional phosphorylation of
pyruvate.

846. Metabolism of lidocaine-C in vitro.
I. C. GeppgEs anp D. E. Dovatas (introduced
by D. L. THomson). Dept. of Anaesthesia and
McGill Univ. Clinic, Montreal General Hosp.,
Montreal, Canada.

Lidocaine (Xylocaine, w-diethylamino-2.6-di-
methylacetanilide) is the most stable local anes-
thetic in general use. There has been no evidence
for hydrolysis of the amide linkage by tissues.
w-bromo-2.6-dimethylacetanilide-C™ was prepared
by known methods from 2.6-dimethylaniline and
bromoagetylbromide-1-C'. Amination of the
w-bromo-2.6-dimethylacetanilid-C' with diethyl-
amine, conversion of the resulting hydrobromide
to the free base, and purification by vacuum sub-
limation gave lidocaine-C" in 19.5% yield based
on the radioacetate employed. Specific activity
was 0.19 mc/mm. Lidocaine-C™ was incubated
with slices of rat cerebral cortex, kidney, and
liver in Krebs’ Ringer-phosphate at 37° for periods
up to 5 hr. CO2 was collected in 20% NaOH.
Carrier lidocaine was added just prior to pre-
cipitation of protein with trichloracetic acid at
the end of the period of incubation. Lidocaine
base was removed by means of a continuous ether
extractor at pH 8. The ether and aqueous phases
were acidified with HCl, dried and made up to
volume in 95% ethanol. Aliquots were plated and
counted in a windowless helium and alcohol flow

FEDERATION PROCEEDINGS

Volume 16

counter. No apparent hydrolysis occurred with
cerebral cortex or kidney slices. With liver, in-
creasing percentages of activity were recovered
from the aqueous phase as the period of incubation
was increased.When a relatively large quantity of
lidocaine-C'* was used, activity was present as
CO2. Gassing with oxygen increased the speed
of hydrolysis. Chromatography and radioautog-
raphy showed that C'-diethylaminoacetic acid
was present as a metabolite; unidentified radio-
active spots were also present on the radio-
autographs.

847. Light-induced oxidation-reduction
changes in Rhodospirillum rubrum ex-
tracts. Davin M. GeELieR* anp Joun D.,
Grecory. Biochemical Research Lab., Massa-
chusetts Gen. Hosp., and Dept. of Biological
Chemistry, Harvard Med. School, Boston.

A photophosphorylation with microsome-like
washed particles from sonic extracts of Rhodo-
sptrillum rubrum was found recently in this labora-
tory (FRENKEL, A., J. Am. Chem. Soc. 76: 5568,
1954). Following up this observation, it has been
found that photophosphorylation depends upon
an inductive reduction, as evidenced by the
marked stimulatory effect of catalytic amounts
(0.1 ug/ml) of lactate or succinate, yielding 3-10
uM ATP/ml. Further stimulation was observed
on addition of phenazine methyl sulfate (0.1
uM/ml), but not by similar dyes. The highest
activity was obtained by a combination of dye
and reductant. With a recording photometer,
optical density changes produced by alternating
light and dark periods were followed. Illumination
of anaerobic particle suspensions containing
reduced phenazine methyl sulfate and succinate
caused an abrupt oxidation of some of the dye,
reaching the new level within 1 sec. Removal of
light then caused a reduction of exactly the
equivalent amount of dye within 5 sec. The pres-
ence of the easily autoxidizable succinate-leuco-
phenazine system precluded the presence of
oxygen. Measurement of the light-dark difference
spectrum of particle suspensions supplemented
with succinate showed the following changes in
the light: 1) increase in absorption around 340 mg
(pyridine nucleotide reduction), 2) decrease at
390 my and increase at 425 my, indicating reduction
of Kamen’s pseudohemoglobin (VERNON AND
Kamen, J. Biol. Chem 211: 648, 1954), 3) increases
at 530 mu and 550-560 my, indicating reduction of
cytochrome components.

848. Light scattering studies on the acto-
myosin-adenosinetriphosphate system. J.
GERGELY AND H. Kouter.* Cardiovascular
Research Lab., Dept. of Medicine, Massachusetts
Gen. Hosp., and Harvard Med. School, Boston.

Per ee eS ee ee eS ee ee ee Ll OU! la

lume 16

d with
rer, in-
-overed
ibation
itity of
sent ag
» speed
autog-
ic acid
radio-
radio-

action
n ex-
in D.
Massa-
logical

ne-like
Rhodo-
abora-
: 5568,
s been
; upon
y the
1ounts
g 3-10
served
e (0.1
ighest
of dye
neter,
rating
ration
1ining
cinate
> dye,
val of
y the
pres-
leuco-
ce of
rence
ented
es in
10 my
se at
ction

AND
eases
on of

icto-
Re: ae
cular
setts

March 1956

Adenosigetriphosphate (ATP) causes dissocia-
tion of actomyosin—natural or reconstituted—
into myosin and actin, and this has been found to
be reflected in light scattering measurements in
the decrease of the weight-average molecular
weight (evaluated according to Zimm) (GERGELY,
J. Biol. Chem., in press). Earlier light scattering
work was done chiefly on natural actomyosin, and
the changes following addition of ATP were
interpreted as deformation rather than dissocia-
tion (BLUM AND Morates, Arch. Biochem.
43: 208, 1953; Tonomura, WATANABE AND YAGI,
J. Biochem. 40: 27, 1953; Buum, Arch. Biochem.
55: 486, 1955). The new evidence has made it pos-
sible to analyze the various phases after addition
of ATP—decrease in turbidity, steady state,
return of turbidity to its original value—in terms
of the specific interaction between myosin and
actin, viz. dissociation and association. Data on
the kinetics and stoichiometry of the various
processes have been obtained by means of an
automatic recording device attached to the light
scattering apparatus. The effect of various factors
on the combination of myosin with actin on the
one hand, and of actomyosin and myosin with
ATP on the other, has been investigated. The
kinetics of the enzymatic splitting of ATP has
been studied optically, and comparisons have been
made with chemically obtained data. A reaction
scheme will be discussed in the light of these
findings.

849. Tyrosine biosynthesis in Escherichia
coli: conversion of prephenic acid (PPA)
to p-hydroxyphenyllactic acid (HPL). Jacat
J. GuosuH (introduced by Bernarp D. Davis).
Pharmacolegy Dept., New York Univ. College of
Medicine, New York City.

PPA is a phenylalanine precursor (through
phenylpyruvate) in EZ. coli (Wetss et al. Science,
119: 774, 1954). Its 4-hydroxy group suggests that
PPA might also lead to tyrosine. To investigate
this possibility without competing conversion
to phenylpyruvate, a mutant (83-5) blocked in
the latter reaction was used. Soluble extracts
catalyze PPA disappearance and the appearance
of a phenolic compound detected by color forma-
tion with diazotized sulfanilic acid or Millon’s
reagent. The enzymatic product, partly purified
by ether extraction and paper chromatography,
resembles synthetic HPL in: 1) chromatographic
behavior in 5 solvent systems (unpublished meth-
ods of Dr. Marvin Armstrong), 2) alkaline vs.
neutral difference spectra and 3) reduction of
DPN in the presence of crystalline lactic dehydro-
genase. Preliminary balance studies indicate
HPL as a major, if not quantitative, product of
PPA in aerobic or anaerobic incubations. HPL
formation is proportional to protein concentra-

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

261

tion; the rate is relatively slow and is not ac-
celerated by a variety of additions. The reaction
is biosynthetically significant since extracts of
two double mutants, blocked in both phenyl-
alanine and tyrosine synthesis, do not convert
PPA to HPL. Although further reactions of added
HPL have not yet been detected in Z. coli extracts,
a material chromatographically consistent with
tyrosine was formed on incubating PPA with
extracts plus cell-residue.

850. Enzymatic mechanisms in the synthesis
of fatty acids. Davin M. Gipson aND MrirIaM
I. Jacosp (introduced by Joun D. Ferry).
Enzyme Inst., Univ. of Wisconsin, Madison.
Beef and pigeon liver have been subfractionated

to yield three soluble enzyme preparations (tenta-

tively designated A, B and C) which on recom-
bination comprise a system for the synthesis of
long-chain fatty acids from acetate (cf. abstract
by Wakil, Porter and Tietz). An examination of
the individual fractions has been undertaken in
order to demonstrate the sequential enzymatic
steps of fatty acid synthesis. Fraction C contains
acetate activating enzyme, 6-hydroxyacyl CoA
dehydrogenase and hydrase. This preparation
catalyzes the oxidation of DPNH on addition of
acetyl CoA at px 6.0. One reaction product is
identical with crotonyl CoA in several paper
chromatographic systems and by analysis with
purified 6-hydroxyacyl CoA dehydrogenase and
hydrase. The enzyme system that performs the
condensation of acetyl CoA with acyl derivatives
of longer chain length also appears to be present
in fraction C. Fraction B catalyzes the reduction
of crotonyl CoA to butyryl CoA in the presence of
TPNH. This reaction may be followed spectro-
photometrically or by determination of the
butyryl CoA formed in the purified acyl CoA
dehydrogenase system. A chromatographic iden-
tification of butyrate is possible in the hydrolyzed
mixture obtained after incubation of radioactive
acetate with fractions B plus C and coenzymes.

Additional evidence will be presented concerning

the integration of these enzymes in the complete

fatty acid synthesizing system.

851. Biosynthesis of diaminopimelic acid.
CuaRLEs GitvarG. Dept. of Biochemistry, New
York Univ. College of Medicine, New York City.
Mutant 26-26 of E. coli, in contrast to the

parental wild type strain, lacks the decarboxylase

which converts diaminopimelic acid to lysine. (B.

D. Davis, Nature, 169: 534, 1952; D. L. Dewry

AND E. Work, Nature, 169: 533, 1952). It was found

that resting cells of the mutant will excrete di-

aminopimelic acid when incubated with glucose

and ammonia. This excretion can be enhanced
four fold by substituting aspartic acid for am-
monia. Homoserine and threonine, known metab-

262 FEDERATION PROCEEDINGS

olites of aspartic acid, are considerably less ef-
fective than aspartic acid or even ammonia. With
aspartic acid as the nitrogen source pyruvate is
almost as active as glucose while alanine and
serine are only 4 and ;'5 as active respectively. It
has also been possible to obtain cell free extracts
from M-26-26 which will synthesize diamino-
pimelic acid. In a study of the requirements of
this enzyme system the following substances have
been found to be stimulatory: pyruvate, aspartate,
glutamate, Mg**, ATP, DPN and TPN. Several
isomers of 8-hydroxydiaminopimelic acid (ob-
tained from D. W. Wooley and J. Stewart) were
tested for their ability to replace diaminopimelic
acid for a mutant which has an early block in the
diaminopimelic pathway. One racemic pair could
replace diaminopimelic acid, three others were
inactive.

852. Uridine diphosphate pentoses in mung
bean seedlings. V. GinsBurG,* P. K. Stumpr
AND W. Z. Hassip. Dept. of Plant Biochemistry,
Univ. of California, Berkeley.

The nucleotides of 4-day-old mung bean seed-
lings were extracted with ethanol and chromato-
graphed on a column of Dowex 1 resin in the
chloride form with increasing concentrations of
hydrochloric acid and sodium chloride. Approxi-
mately 2.7 mm of nucleotides were obtained from 10
kg of seedlings, half of which were derivatives of
uridine. A fraction of these nucleotides were found
to possess the properties associated with uridine
diphosphate glucose. This fraction on hydrolysis
with 0.01N hydrochloric acid for 10 min. yielded
uridine diphosphate together with the sugars
glucose, galactose, xylose, and arabinose.

853. Specificity of peptide moieties of the
lipoproteins of human plasma. Davip
Gitiin, Davip G. CoRNWELL AND J. L. ONCLEY.
Depts. of Pediatrics and Biological Chemistry,
Harvagl Med. School, and Children’s Med. Center,
Boston, Mass.

In this study, the evidence obtained clearly
indicates that the peptide moieties of the lipopro-
teins of human plasma are not identical. The
moieties are specific not only for each of the major
electrophoretic classes of lipoproteins, a- and
B-lipoproteins, but also specific, at least, for each
of the density classes of 8-lipoproteins studied:
Sr 3-8, S- 15-400 and the chylomicra. It has been
demonstrated that lipoproteins labelled in the
peptide moiety with I'*! and incubated with whole
plasma or whole £-lipoprotein fractions in vitro,
or after intravenous injection into humans, are
isolated only with the homologous ultracentrifugal
or electrophoretic class of unlabelled lipoprotein.
There is no apparent exchange of labelled peptide
moieties among lipoproteins of different density

Volume 1§

or electrophoretic classes. Immunochemically, the
a- and 8-lipoproteins are easily distinguished and
the precipitation curves of the -lipoproteing
differ for the different classes studied. Meta-
bolically, the behavior of labelled a-lipoprotein
differs from that of the 6-lipoproteins, among
other things, having a normal half-life of 4 days
as compared to 3 days for the latter. The metabolic
behavior of different density classes of labelled
8-lipoproteins also differ from each other.

854. Electron transferring particle. J. L,
GLENN AND F. L. Crane (introduced by C. A,
ELVEHJEM). Inst. for Enzyme Research, Univ. of
Wisconsin, Madison.

An electron transferring particle (ETP) which
transfers electrons from succinate and DPNH to
oxygen has been isolated from beef heart mito-
chondria. At 38°C the maximum rate of succinate
and DPNH oxidation is 2.0 and 3.8 umM/min/mg
protein respectively, which is 5-fold that of mito-
chondria. Phosphate is required for maximum
oxidation and metal binding agents cause further
stimulation, whereas antimycin a inhibits. ETP
has constant composition, with flavin, heme, non-
heme iron, and copper in a ratio of 1:8:40:8,
Alcohol-extractable lipid constitutes approxi-
mately 30% of dry weight. After reduction the
absorption spectrum shows peaks at 603, 562, 552,
and 525 my and Soret bands with a peak at 428 my
and shoulders at 417, 420, and 444 my, which indi-
cate the presence of cytochromes b, c, ci, and a.
An ETP preparation containing high levels of
cytochrome c can be obtained, but there is no
consistent relationship between the cytochrome
c content and activity. ETP can be broken into
fragments, each representing part of the original
activity. Particles corresponding to the succinic
dehydrogenase complex (D. E. Gresn, et al., J.
Biol. Chem. 217 : 551, 1955), DPNH and cytochrome
oxidases (B. MackuEr, Federation Proc. 14: 802,
1955), and the soluble succinic and DPNH de-
hydrogenases (R. BAsForRD AND B. DE BERNARD,
see abstract) have been derived from ETP.

855. Biochemical study of HeLa cells. Howarp
GoLpFINE,* Ray KoppetMan* anv E. A.
Evans, Jr. Dept. of Biochemistry, Univ. of
Chicago, Chicago, Ill.

Strain HeLa, human cervical carcinoma cells
were grown as described by Syverton etal. (J. Lab.
& Clin. Med. 43: 286, 1954) using Eagle’s medium
(Science 122: 501, 1955). The dry weight of the
cells is 0.65 mg/10® cells. The total phosphorus is
25 y/10® cells. The acid-soluble phosphorus frac-
tion is 45% of the total, the alcohol-ether soluble
fraction is 20-25%, and the protein phosphorus
fraction is 5%. Nucleic acid contributes 25% of
the total phosphorus. The ratio RNA:DNA is 2:1.

ae ee ee Ne eee ae ee

ume 1§

ly, the
ed and
roteing
Meta-
»rotein
among
4 days
tabolie
ibelled

J. 8
CLA,
niv. of

which
NH to
mito-
cinate
in/mg
‘mnito-
imum
urther
_ ETP
, non-
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March 1956

Chromatographic analysis shows thymine present
in the DNA, uracil present in the RNA, and
guanine, adenine and cytosine in both types of
nucleic acids. Metabolic studies were carried out
in the Warburg respirometer on washed cell
preparations. In the absence of added substrate
the mean Qo, is 8 and the mean R.Q. is 0.73. In
the presence of glucose, acid production in bi-
carbonate buffer in N»-CO2 is high, with lactic
acid apparently the chief product. In the presence
of glucose, acid production in Ringer-phosphate
buffer in air is also high. The oxygen uptake of
washed cells was followed for 4 hr. in the presence
of added substrate. Pyruvate, butyrate, D-ribose,
a-alanine, malate, and glutamate had little or no
effect. Lactate, succinate, oxalacetate, a-keto-
glutarate, citrate and aspartate maintained
oxygen uptake at levels above that of cells respir-
ing in the absence of added substrate.

856. Citrate-condensing enzyme of mycobac-
terium tuberculosis. Dexter S. GoLpMAN.
VA Hosp., Madison, Wis.

A soluble enzyme catalyzing the synthesis of
citrate from acetyl-CoA and oxalacetate has been
found in cell-free extracts of M. tuberculosis,
H37Ra. This enzyme appears to be very similar
to that of animal cells. When the bacterial citrate-
condensing enzyme is coupled with phosphotrans-
acetylase (Cl. kluyvert or M. tuberculosis) and malic
dehydrogenase (M. tuberculosis) the following re-
action sequence is observed: 1) Acetyl-P + CoA=
acetyl-CoA + P; 2) L-Malate + DPNt = oxal-
acetate + DPNH + Ht; 3) Oxalacetate + acetyl-
CoA — citrate + CoA. Cell-free extracts of M.
tuberculosis are prepared by sonic oscillation. 1.0
mg of the crude extract protein catalyzes the
formation of 0.3 um of citrate in 5 min. at 30°. By
fractionation with ammonium sulfate and elution
from calcium phosphate gel the specific activity
is raised to about 5.

857. 5-Phosphoribosylamine, a precursor of
glycinamide ribotide. Davin A. GoLpTHWAIT
(introduced by G. R. Greensera). Dept. of
Biochemistry, Western Reserve Univ. Med. School,
Cleveland, Ohio.
5-Phosphoribosylpyrophosphate (PRPP), gluta-

mine, glycine and ATP were required in a pigeon

liver extract for the synthesis of glycinamide

ribotide (GoLpTHWAIT, GREENBERG AND P#a-

Bopy. Biochim. et biophys. acta 18: 148, 1955).

PRPP reacted with glutamine, and while no

product accumulated, a balance study suggested

that 5-phosphoribosylamine (PRA) might be
formed. This compound, synthesized chemically,
could replace PRPP and glutamine in the syn-
thesis of glycinamide ribotide. ATP was required
for the enzymatic synthesis of glycinamide ribo-

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

263

tide from PRA and glycine. A small exchange of
orthophosphate with ATP occurred only in the
presence of both glycine and PRA. A glycine-
catalyzed exchange of pyrophosphate did not
occur. An hydroxamic acid was formed when
glycine, ATP, and hydroxylamine were incubated
with the enzyme fraction. PRA was unstable in
acid and was rapidly degraded to ribose-5-phos-
phate. It retained 97% of its precursor activity
when kept in 0.02 m KOH at 3° for 18 hr., but only
7% at pu 7.0. A hexosiminic acid phosphate
(HAP) derivative of PRA, synthesized by HCN
addition, was purified on a Dowex-l-column. On
treatment with ninhydrin, HAP yielded a com-
pound which reacted with orcinol. Enzymatically
dephosphorylated HAP migrated in 4 solvents
identically with the hexosiminic acid derivative
of ribosylamine. Acetylation of PRA with acetic
anhydride was unsuccessful.

858. Formation of ribonucleic acid by pigeon
liver homogenates. EuGENE GOLDWASSER
(introduced by ALBERT DoRFMAN). Argonne
Cancer Research Hosp., Univ. of Chicago, Chi-
cago, Til.

Some aspects of the biosynthesis of ribonucleic
acid (RNA) by cell-free preparations of pigeon
liver homogenate have been studied using either
adenine-8-C' or labeled adenosine-5’-phosphate
(AMP) as precursors. Experiments using adenine
as the precursor and attempting to dilute its in-
corporation into RNA by adenosine, adenosine-
2’(3’)-phosphate or AMP yielded RNA samples
with specific activities not appreciably different
from the control. However, AMP labeled in either
the adenine moiety with C" or with P®* has been
shown to be a precursor of RNA. These data sug-
gest that the pathway of incorporation of adenine
into RNA may not involve AMP. Adenine may
possibly form ADP without either adenosine or
AMP as obligate intermediates and ADP may be
used for RNA formation. Alternatively, adenine
may enter an already formed polyribophosphate
backbone by an exchange process. The distribu-
tion of adenine-8-C" in the RNA of the subcellular
components after incubation of the whole ho-
mogenate with adenine indicates a different
situation from that observed with whole animal
studies. The specific activities of the RNA of the
‘nuclear’ fraction, mitochondria, microsomes and
soluble fraction were the ratios of 1.4:1.0:1.1:6.5,
respectively.

859. Reaction of cyanide with cysteine and
other sulfhydryl compounds. Jutius Gouv-
Bow,* RaupH K. Barciay anp C. CHESTER
Srocx. Sloan-Kettering Inst. for Cancer Research
and Sloan-Kettering Div., Cornell Univ. Grad.
School of Med. Sciences, New York City.

The reaction of cyanide with cysteine at alkaline

264

pus liberates ammonia and forms a ninhydrin-
reactive compound. This compound has been
tentatively identified as 4-thiazolidinecarboxylic
acid. Studies on the specificity of this reaction
indicate that a free sulfhydryl] group is necessary,
inasmuch as cysteine, homocysteine, reduced
glutathione and penicillamine react, while S-sub-
stituted compounds such as cystine, S-methyl-
cysteine, S-ethylcysteine, cysteic acid and me-
thionine do not react to liberate ammonia. Some
of the nitrogen liberated apparently is derived
from the cyanide, since thioglycolic acid also
reacts with cyanide to liberate ammonia. The
cyanide must be in the ionic form, since methyl
cyanide is not reactive. In studies with animals, a
lethal dose of cyanide was found to be nontoxic
when administered to rats previously treated with
cysteine. 4-Thiazolidinecarboxylic acid was de-
tected in the urine of animals treated in this
manner. Increased doses of cyanide resulted in
increased urinary excretion of 4-thiazolidine-
carboxylic acid. These studies offer strong evi-
dence that 4-thiazolidinecarboxylic acid is a result
of the detoxification of cyanide by cysteine.

860. New pyrimidine nucleoside analogues.
Irvine GoopmMaNn. Wellcome Research Labs.,
Tuckahoe, New York.

The glycosyl ureas have been known for over
half a century and have been the subject of
numerous chemical and biological investigations.
Bergmann and Johnson (J. Am. Chem. Soc. 60:
1916, 1938), in a study of glycosyl ureas, attempted
to apply the Traube synthesis of 6-aminopyrimi-
dines (Ber. 33: 1371, 1900) to the synthesis of
pyrimidine nucleosides, but were unable to obtain
the desired products by this method. The present
report deals with the synthesis of 1-glycosyl-6-
aminouracils by the Traube reaction. The nucleo-
sides were obtained by treating 2,3 ,4,6-tetraacet-
ylglucosyl-l-urea, for example, with cyanacetic
acid, and effecting ring closure by means of
dilute alfali to form 1-(2’ ,3’,4’,6’-tetraacety]-
glucosyl)-6-aminouracil. The products may be
deacetylated by ethanolic NH; to yield the free
nucleosides. A variety of 1-substituted alkyl-,
aryl- and heterocyclic 6-aminouracils were pre-
pared in this study and their physical and chemical
properties were investigated. The region of
maximum ultraviolet absorption for this series is
about 265 my at px values from 1 to 11.

861. Responses of the respiratory system of
Azotobacter in the presence of sodium
azide. C. R. Goucner* anp W. Kocuo.arty.
Army Med. Research Lab., Fort Knox, Ky.
Reflectance spectra of cells of Azotobacter ‘‘Q”

(J. Biol. Chem. 211: 613, 1954) in the presence of

exogenous substrate and of O2 or Nz appear identi-

FEDERATION PROCEEDINGS

Volume 18

cal when judged by spectrophotometric analysis
in the integrating sphere (J. Optic. Soc. America
45: 460, 1955). Therefore, it may be inferred that
the cytochromes of this organism are reduced at a
more rapid rate than they are oxidized. Thus, in-
hibition of the rate-limiting activity of the cyto-
chrome oxidase would result in an inhibition of
those activities which usually depend upon the
function of this enzyme, such as the Nadi reaction
and the uptake of oxygen. Azotobacter cells do not
show a reduced cytochrome spectrum in the ab-
sence of exogenous substrate and in the presence
of oxygen. Such cells, however, in the presence of
10-* m azide and oxygen show a reduced cyto-
chrome spectrum, indicating that sodium azide
has retarded the rate of the oxidation of the
cytochromes reduced by endogenous material.
Azide in 10-* molar concentration prevents the
Nadi reaction of whole cells without significantly
affecting the rate of acetate oxidation; with cell
extracts the oxidation of succinate is inhibited
only slightly by 10-2 m azide. These observations
suggest that the indophenol oxidase system and/or
the cytochrome oxidase system of this Azotobacter
may be inoperative in the oxidation of acetate in
the presence of sodium azide.

862. Effect of x-irradiation on hepatic choles-
terol synthesis. R. Gorpon Goutp, L. Vir-
q@intA Lotz* anp Epitx M. Litiy.* Los Alamos
Scientific Lab., Univ. of California, Los Alamos,
N.M.

Whole body x-irradiation has been found to
result in a pronounced increase in the rate of
hepatic cholesterol synthesis in rats as estimated
from the incorporation of 1-C'4-acetate. Groups of
irradiated and control rats were fasted for 24 or
48 hr., C14-acetate doses proportional to body
weight given by intraperitoneal injection, and the
animals killed 4 hr. later. The C'4-uptake was de-
termined in the cholesterol and fatty acids of
liver, adrenals, certain other tissues and residual
carcass. Cholesterol specific activity values were
20-30 times as high in livers of rats 48 hr. after
2400 r as in controls; 24 hr. after the same dose,
the effect was a 5-10 fold increase; and 48 hr. after
1200 r it was 10-15 fold. The irradiated animals
showed a relative increase in liver weight (ex-
pressed as percentage of body weight) of 33% at
24 hr. and 42% at 48 hr., as compared with fasted
controls; cholesterol concentrations in liver were
slightly lower in irradiated animals. This radiation
effect appeared to be independent of the route of
administration or amount of acetate given, indi-
cating a true change in synthetic rate. Changes in
the rates of fatty acid synthesis in liver and in
cholesterol synthesis in most extra-hepatic tissues
and in residual carcass were either much smaller
or not significant. Adrenal glands, however,

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March 1956

showed approximately as large an increase in
cholesterol synthesis following irradiation as did
liver. (Work done under the auspices of the
Atomic Energy Commission.)

$63. Biochemical response to trauma. V.
glutamine, glutamic acid, ammonia in the
brain. Irvine Gray, JoHN M. JOHNSTON AND
Cecit1a W. SpEARING (introduced by Exiza-
petH R. B. Smitu). Walter Reed Inst. of Re-
search, Washington, D.C.

Male Sprague-Dawley rats (Holtzman Farms),
925-275 gm, were tumbled for 600 turns in a
Noble-Collip drum. Mortality was 90-100%. Total
glutamine and glutamic acid was measured
manometrically using Cl. welchii to decarboxyl-
ate. Ammonia of the butyramide, formed from
glutamine as a result of the decarboxylation, was
determined after liberation by K:CO;. This was a
measure of the glutamine. Ammonia content of
the brain was determined by difference after cor-
recting for all controls. The concentration of these
compounds remained unchanged in the brain im-
mediately following trauma. The level of gluta-
mine was increased at 30 min. after the trauma
but at 60 min. had increased no further. The am-
monia concentration, which was not different
from the control values immediately or 30 min.
after the trauma, had become significantly ele-
vated at the 60-min. interval. The significance of
the data is discussed in light of the toxicity of
ammonia and the function of the reaction, glu-
tamic acid + NH; — glutamine, in brain me-
tabolism.

864. Synthesis and metabolic properties of
dihydropyrimidine nucleosides. MaAvurRIcE
GREEN,* JANET LICHTENSTEIN,* HAzEL
BARNER* AND Seymour 8. Cowen. Children’s
Hosp. of Philadelphia and Univ. of Pennsylvania
School of Medicine, Philadelphia.

As part of an investigation of DNA pyrimidine
biosynthesis in growing and virus-infected bac-
teria, the role of dihydropyrimidine derivatives
was examined. Dihydrouracil, dihydrothymine,
dihydrothymidine, dihydrodesoxyuridine, and di-
hydrouridine were synthesized by catalytic
hydrogenation with yields of about 90%. All but
dihydrouridine were obtained as crystalline solids;
all were chromatographically homogeneous. Di-
hydrothymidine, dihydrodesoxyuridine, and di-
hydrouridine were readily hydrolyzed under the
acid conditions of the Dische diphenylamine and
the Bial orcinol reactions to give quantitative
color values for desoxypentose and pentose. The
dihydropyrimidine nucleosides were split in dilute
acid at 100° to yield the corresponding free di-
hydropyrimidines. Cell-free extracts of E. coli,
which were highly active in the arsenolysis and

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

265

phosphorolysis of thymidine, desoxyuridine, and
uridine, did not cleave dihydropyrimidine nucleo-
sides. The dihydropyrimidines were inactive in
transglycosidation reactions with thymidine
under conditions in which desoxyuridine was
readily formed from uracil and thymidine. How-
ever, the enzymatic cleavage of uracil and thymine
nucleosides was partially inhibited by several of
the dihydropyrimidine derivatives. Dihydrouracil
and its nucleosides did not support the growth of,
or bacterial virus multiplication in, uracil-requir-
ing strains of EZ. coli; dihydrothymine and di-
hydrothymidine did not support the growth of a
thymine-requiring strain. Dihydrothymine in-
hibited the growth of the uracil- and thymine-
requiring strains at 10 X the molar concentration
of uracil and thymine respectively, while dihydro-
thymidine was only slightly inhibitory. Dihydro-
uracil and derivatives were not inhibitory at these
levels.

865. Riboflavin deficiency in the rhesus
monkey. Louis D. GREENBERG AND JAMES F.
Rinenart.! Dept. of Pathology, Univ. of Cali-
fornia School of Medicine, San Francisco.
Riboflavin deficiency was studied in 6 rhesus

monkeys maintained on a basal diet of sucrose,

corn oil, salts, and alpha-protein supplemented
with 0.3% pu-methionine, and all the vitamins
known to be essential for the monkey with the
exception of riboflavin. Two animals which had
received riboflavin throughout the experiment
served as controls. First, the monkeys were per-
mitted to remain on the diet with an adequate
supply of riboflavin for approximately 4-8 mo. in
order to obtain control data. Growth on this diet
was only slightly inferior to that which has been
observed on an 18% casein diet. The first de-
ficiency manifestations, consisting of periorbital
dermatitis or scaliness and dermatitis around the
mouth (cheilosis) appeared 5-11 wk. after with-
drawal of the vitamin. This was followed fre-
quently by the appearance of dermatitis at other
sites, namely, toes, arms, and abdomen. In addi-
tion, the monkeys developed hypochromic anemia
which was slow to respond to riboflavin therapy.
The total serum riboflavin and total cholesterol
which were followed throughout the control and
deficient periods were observed to fall markedly
in a more or less parallel fashion during the
periods of deprivation and to increase in a similar
manner following restoration of riboflavin to the
diet. (Supported in part by a grant from the
Natl. Vitamin Fndn.)

866. Reduction and hydrolysis of disulfide
bonds in a-chymotrypsin. Eston M. Gross
AND RicHarD E@an (introduced by Wiuu1aM H.

1 Deceased.

266

SummMeERson). Enzyme Chemistry Branch, Chemi-

cal Corps. Med. Labs., Army Chemical Center, Md.

Complete reduction of the disulfide bonds in
a-chymotrypsin (ChTr) by potassium sulfite can
be accomplished in the presence of a 6-molar
excess of p-chloromercuribenzoate (PCMB), an
agent which reacts with the SH groups as they are
formed. Analysis for SH, performed by back-
titrating the excess standard PCMB with stand-
ard cysteine solution and nitroprusside as indi-
cator, showed 4.2+0.2 cysteine residues per mole
of reduced ChTr (corresponding to the same
number of original S—S bonds) using a molecular
weight of 22,000. This compares with the value of
4.2 cysteine residues per mole, as calculated from
Brand’s corrected analytical data for total cystine,
using the same molecular weight. The completely
reduced ChTr was shown to be devoid of esteratic
activity. Reduction also produced a significant
breakdown of the protein structure. Seventy %
of the product was soluble in 20% trichloroacetic
acid and passed through a dialysis membrane.
Enzymatic activity was lost more rapidly if the
protein concentration was above 5 mg/ml. At
greater concentrations, molecular aggregation
accelerated activity loss. Structural breakdown
could also be induced in the absence of reducing
agent by allowing the protein to remain in PCMB
solution overnight. This reaction, much slower
than reduction, is ascribed to a hydrolysis of S—S
bonds catalyzed by the PCMB removal of SH
from the equilibrium. The heterogeneity of the
product was illustrated by sedimentation be-
havior.

867. Mechanism of DPNase catalyzed ex-
change reactions. LAWRENCE GROSSMAN
(introduced by NaTHan O. Kaptan). McCollum-
Pratt Inst., Johns Hopkins Univ., Baltimore, Md.
A general survey of enzymes present in human

and bovine erythrocytes capable of acting on the

high egergy linkage between the pyridine nitrogen
of the nicotinamide and carbon one of the ribose
of DPN, nicotinamide mononucleotide (NMN)
and nucleoside (NR) has been carried out. The
sensitivity of these enzymes to nicotinamide and
their ability to catalyze exchange reactions have
been investigated. There is evidence to indicate
that three different enzymes are acting on DPN,
NMN and NR. Purification of these enzymes by
fractional ammonium sulfate precipitation and
other procedures results in a loss of nicotinamide
sensitivity of the nicotinamide ribosidase. Nico-
tinamide sensitivity of the ribosidase was restored
by the addition of a second fraction. The nico-
tinamide-insensitive Neurospora DPNase was
transformed to a ‘sensitive’ enzyme upon addition
of this same fraction which can be obtained from
either bovine or human hemolysates. The ‘sensi-

FEDERATION PROCEEDINGS

Volume if

tive’ Neurospora DPNase can catalyze an ex.
change reaction between the nicotinamide moiety
of DPN and 6-C!4-nicotinamide. Similar nieo.
tinamide ‘sensitizing’ fractions can be obtained
from the beef spleen DPNase. The significance of
these fractions which can promote nicotinamide
sensitivity will be discussed with respect to the
mechanism for conserving the high energy bond
of the nicotinamide riboside linkage.

868. Metabolism of fluorene derivatives by rat
liver slices. Hetmut R. GutTmMann, Joun 4,
Prerers* anD Hersert T. Nacasawa.* Radio.
isotope Service, VA Hosp. and Dept. of Physio.
logical Chemistry, Univ. of Minnesota, Minne.
apolis.

Previous experiments on the in vitro metabolism
of the carcinogen 2-acetylaminofluorene suggested
that an oxidized metabolite was irreversibly
bound to rat liver proteins (J. Biol. Chem. 211: 63,
1954). Further studies have been carried out to
determine whether hydroxylation of the sub-
strate is necessary for protein binding. 8% of
2-acetylaminofluorene-9-C!4 was oxidized to
2-hydroxy-7-acetylaminofluorene-9-C!4 by rat
liver slices and 2% of the substrate radioactivity
was bound to liver proteins. Similar data for
hydroxylation and protein binding were obtained
when 2-benzoylaminofluorene-9-C!4, a weak car-
cinogen, was the substrate. In contrast, only 2%
of 2-acetylaminofluorene-9-C'* was oxidized and
0.6% was bound by rat liver homogenates. Heat-
ing of liver slices or incubation at px 2 abolished
protein binding and a 77% reduction was ob-
served in the presence of cyanide. In these experi-
ments, no hydroxylation was detectable by
chromatography. These data indicate that sub-
strate oxidation is required for protein binding.
Formation of 2-aminofluorene appears to have no
relation to protein binding since 2-aminofluorene-
9-C!4, 2-acetylaminofluorene-9-C!4 and 2-benzoyl-
aminofluorene-9-C!4 were bound to the same
extent. In contrast, the extent of in vitro hydroly-
sis of the labeled acetyl and benzoyl compounds
was 20 and 2% respectively. Previous experiments
suggested that 2-aminofluorene might act as &
high-energy acetyl trap (J. Biol. Chem. 216: 713,
1955). The possibility was tested that succinate
might acylate 2-aminofluorene in a manner simi-
lar to acetate. 0.9% of N-(2-fluorenyl-9-C"*)-sue-
cinamate was isolated following incubation of
2-aminofluorene-9-C!* and succinate in the pres-
ence or absence of liver slices.

869. Stimulation of carbamyl phosphate syn-
thesis by a factor isolated from liver. Lz0
M. Haui* anp Puiuip P. Cowen. Dept. of
Physiological Chemistry, Univ. of Wisconsin,
Madison.

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OHN HH,
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abolism
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211: 63,
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. Heat-
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March 1956

Optimum. conditions for the biosynthesis of
carbamyl phosphate from ammonia and carbon
dioxide by mammalian liver enzymes require
ATP, Mg**, and catalytic amounts of certain
derivatives of glutamic acid (Biochim. et biophys.
acta. 17: 279, 1955). In an enzyme system lacking a
glutamic acid derivative, a small but significant
synthesis of carbamyl phosphate occurs using rat
liver mitochondrial acetone powder as the enzyme
source. A factor has been partially purified from
an extract of rat liver mitochondrial acetone
powder which stimulates the synthesis of carbamyl
phosphate in a system free of added glutamyl
derivatives. The factor has been purified by
chromatography on Dowex-2-formate, and moves
with an Rr of 0.23 in a solvent system containing
0.1 m phosphate pu 6.8/ammonium sulphate/n-
propanol, 100/60/2. At the present stage of puri-
fication, an aqueous solution of the factor, px 5,
exhibits an absorption maximum at 260 my. The
relationship of the absorption properties and the
activity in addition to the characterization of the
factor is at present under study.

870. Nature of yeast cephalins. Donaup J.
HANAHAN AND Dovuauas N. Ruopss.* Dept. of
Biochemistry, Univ. of Washington, Seattle, and
Low Temperature Research Station, Cambridge,
England.

The cephalins of bakers’ yeast have been frac-
tionated in part by means of silicic acid chroma-
tography into 3 major components. In the pro-
cedure selected for general separations, the alcohol
insoluble, noncholine containing phospholipides of
bakers’ yeast were adsorbed from CHCl;-MeOH
(4:1) on a silicic acid column. Elution with the
same solvent removed 55-60% of the applied
phosphorus in 2 rather well defined bands; Ist, a
fast running fraction of N/P, 0.20 and 2nd, a
slower moving component of N/P, 0.90-1.0. When
the eluting solvent was changed to CHCl;-MeOH
(1:1), the remainder of the phosphorus could be
removed as an apparent single component of N/P,
0.33. This latter substance (BF) was a stable,
light tan powder which was soluble in CHCl;,
glacial acetic acid and benzene. Analysis showed it
to have the following composition: %P, 3.60;
%N, 0.44; [al + 10.8°; %F.A., 47.5; F.A./P, 1.3.
Hydrolysis in 6N HCl at reflux for 5 hr. resulted
in the liberation of 65% of the phosphorus as in-
organic phosphorus. In addition, at least one
amino acid is present. No sphingosine, inositol,
carbohydrate or sterol have been detected in this
fraction. The possible structure of BF will be dis-
cussed. Of added interest is the observation that
the original pre-column cephalin fraction con-
tained approximately 3% of a,a’ trehalose in the
free form and also detectable amounts of free
amino acids.

' ave tae

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

267

871. Microbial studies of 6-azuracil, an in-
hibitor of uracil. R. E. HanpscuuMacHER*
AND ARNOLD D., We tcu. Dept. of Pharmacology,
Yale Univ., New Haven, Conn.

Studies have been made of 6-azuracil (AzU)
(s-triazine-3,5-dione) as an inhibitor of growth of
microorganisms with different requirements for
exogenous uracil (U). With a strain of Lactobacillus
bulgaricus 09 able to utilize U for growth, AzU
was a competitive antagonist of U and a non-
competitive antagonist of orotic acid; when added
during the phase of exponential growth AzU
caused an inhibition which was not reversed by
U, uridine or uridine-5’-phosphate added 2 hr.
subsequently. Growth of Leuconostoc citrovorum
(Pediococcus cerevisiae), on thymidine or leuco-
vorin, and of Lactobacillus leichmanti, on thymi-
dine or vitamin Biz, was inhibited strongly by
AzU in the absence of U; these inhibitions were
prevented by the presence of excess U. Strepto-
coccus faecalis, which does not require exogenous
U, was inhibited by AzU when grown on thymine,
thymidine or pteroylglutamic acid (PGA). Added
at the time of inoculation, AzU was very much
more inhibitory in the presence of low levels of U
than in its absence (high levels of U nullified the
action of AzU); those organisms which grew in the
absence of U were resistant to AzU. However,
when added during exponential growth on thy-
mine, AzU was most inhibitory in the absence of
added U and resistant strains did not emerge.
Supraoptimal, as compared to optimal, amounts
of thymine, thymidine or PGA increased the de-
gree of inhibition caused by AzU. Resting cells of
S. faecalis incubated with thymidine and AzU
accumulated a compound which had the properties
of AzU deoxyriboside.

872. Metabolism of C'‘-carboxyl-labeled 3-
hydroxyanthranilic acid in the rat. L. V.
HANKEs AND L. M. HenpErson. Medical Dept.,
Brookhaven Natl. Lab., Upton, N. Y., and Dept.
of Chemistry and Chemical Engineering, Univ. of
Illinois, Urbana.

Carboxyl-labeled 3-hydroxyanthranilic acid
synthesized according to the method of Ciereszko
and Hankes (J. Am. Chem. Soc. 76: 2550, 1954)
was injected into male adult rats at the levels of
3.1 and 19.1 mg. The rats were housed in a glass
apparatus for expired air and urine collection.
Quinolinic acid was isolated from the urine of the
rat that received 19.1 mg. The 3-hydroxyanthra-
nilic acid was metabolized very rapidly to carbon
dioxide. Of the administered C'4 approximately
60% appeared in the CO: of the expired air within
3 hr., and nearly 90% appeared in the expired air
and urine within 12 hr. The rapid oxidative
metabolism of this compound would account for
its relative ineffectiveness as a substitute for

268

nicotinic acid in the rat. The quinolinic acid
excreted was about 30 times as much as that
excreted by control rats. The C4 per molecule of
the quinolinic acid was approximately 0.91 that of
the administered 3-hydroxyanthranilic acid, indi-
cating direct conversion of 3-hydroxyanthranilic
acid to quinolinic acid. Decarboxylation of the
isolated quinolinic acid in the alpha-carboxyl
position by heating to 220° (J. Biol. Chem. 211:
725, 1954) and measurement of the C" activity of
the carbon dioxide liberated showed relatively no
activity in this position; the C14 was in other parts
of the quinolinic acid molecule. Work is in progress
to determine the location of the C!*. (Supported by
the Atomic Energy Commission.)

873. Isolation of an adenosine nucleotide
from liver. R. G. Hansen, EvizaBetH HaGe-
MAN,* R. A. FREEDLAND* AND D. R. WILKIN.*
Lab. of Biochemistry, Dept. of Dairy Science,
Univ. of Illinois, Urbana.

Two separate extraction procedures have been
employed to isolate nucleotides from the livers of
chicks, rats and guinea pigs. Immediately follow-
ing decapitation of the animals, the livers were
removed and homogenized either in cold 0.6 N
perchloric acid or 60% ethanol. Separation of the
nucleotides was accomplished with an ion ex-
change column. Purification of one of the adenine
containing fractions was achieved by chroma-
tography, first, on Dowex-l-formate, then on a
mixed bed of charcoal and cellulose. One hundred
grams of fresh liver yielded about 10 um of nucleo-
tide with either extracting solvent. The ultra-
violet spectrum of the purified nucleotide was
identical with adenine. The hydrolyzed nucleotide
yielded theoretical quantities of uric acid when
treated with adenine deaminase then xanthine
oxidase, and 2,8-dihydroxy adenine when treated
with xanthine oxidase alone. The molecular ratio
of adenine to ribose and phosphate was 1:2:2.
Both phosphates were acid stable. Alkaline hy-
drolyzates of the nucleotide were deaminated by
adenosine deaminase. Only the ribose-5-phosphate
isomer was detected in acid and Dowex-1 catalyzed
hydrolyzates of the nucleotide. The nucleotide
yielded adenosine mono- and di-phosphates upon
mild alkaline hydrolysis. Since DPN was not de-
composed when subjected to the same treatment
as the liver, the nucleotide did not result from
hydrolysis of DPN during the isolation pro-
cedure. These data support the conclusion that
the nucleotide isolated was adenosine-diphos-
phate-ribose.

874. Effect of calcium ions and ethylene-
diamine tetraacetate (EDTA) on myosin
B-adenosinetriphosphatase. N. R. Hans
(introduced by E. M. Brings). Univ. of Buffalo,
Chronic Disease Research Inst., Buffalo, N. Y.

FEDERATION PROCEEDINGS

Volume §

The action of EDTA and calcium, both aetj.
vators of the Myosin B ATPase-ATP system have
been compared. Others have demonstrated that
there is a relationship between KCl levels and the
degree of Na-EDTA activation. In our experi.
ments the activation of the system by calcium
decreases with increasing ionic strength of KC,
This contrasts with the action of EDTA. At all
levels of KCl studied, an inactivation of Myosin
B-ATPase was found for the same respective Ca
concentrations. The inhibitory action of EDTA
in low concentrations therefore cannot be due to
its chelating action on Ca but rather to a compe.
tition with another element. EDTA stimulation
also appears to be competitively inhibited by Ca.
Correlating the above findings evidence is pre-
sented that a) Ca replaces the natural activator at
the enzyme surface. The resulting complex is in-
ferior to the original one. (Inhibition of the
system by low Ca concentrations.) b) Potentially
active groups combine with Ca at high Ca levels,
(Activation at high Ca concentrations.) c) EDTA
acts at the same site of the enzyme surface as Ca,
(Inhibition or stimulation depending upon the
individual concentration levels. Competitive
inhibition between Ca and EDTA.) d) An en-
zyme-Ca complex forms first and this complex
combines through Ca with the substrate. (Inhibi-
tion over the same range of low Ca concentrations
regardless of ionic strength of KCl. Decreased
activation with increasing KCl concentration.)

875. Bacterial fermentation of nicotinic acid,
Isaac Harary (introduced by J. SNokg).
Radioisotope Service, VA Hosp., Long Beach,
and Dept. of Physiological Chemistry, School of
Medicine, Univ. of California, Los Angeles.

An anaerobic spore forming rod has been iso-
lated from Potomac river mud which ferments
nicotinic acid. In the presence of peptone and
yeast extract no growth occurred unless a high
level of nicotinic acid was included. The bacteria
will not ferment picolinic, isonicotinic, quinolinie,
isocinchomeronic, anthranilic, 3-hydroxy-anthra-
nilic, para-amino benzoic and kynurenic acids or
pyridine and methyl nicotinamide. Nicotinamide,
on the other hand, was as actively fermented a8
nicotinic acid. Balance experiments with dried
cell preparations in phosphate or bicarbonate
buffers at pu 7.4 indicated that for each mole of
nicotinic acid disappearing 1 m each of propioni¢
acid, acetic acid, CO. and NH; was formed 38
represented by the following equation: 4H.O +
nicotinic acid > acetic acid + propionic acid +
NH; + CO:. The steam volatile acids were deter-
mined by Duclaux distillation and separation on
paper chromatograms and on silica gel columns.
When equimolar amounts of nicotinic acid and
methylene blue were incubated anaerobically with

olume if

th acti.
em haye
ted that
and the
- experi.
calcium
of KCl,
.. At all
Myosin
stive Cg
’ EDTA
e due to
, compe-
nulation
1 by Ca,
is pre-
vator at
ex is in-

of the
entially
a levels,
) EDTA
e as Ca,
pon. the
petitive
An en-
-omplex
(Inhibi-
trations
creased
ation.)

ic acid,
SNOKE),
Beach,
chool of
8.
en. isd
rments
ne and
a high
acteria
nolinie,
anthra-
cids or
amide,
nted as
. dried
bonate
nole of
opionie
ned a8
H2O +
acid +
deter-
ion on
lumns.
id and
ly with

March 1956

a suspension of dried cells all of the methylene
blue was reduced. Paper chromatography of the
reaction mixture demonstrated that all of the
nicotinic acid had disappeared and a new com-
pound with a lower Rf was formed. This com-
pound contained an acidic group and exhibited
fluorescence similar to that of nicotinic acid. On
the basis of its chromatographic behavior and
ultraviolet absorption spectrum, the compound
has been tentatively identified as 6-hydroxy-
nicotinic acid.

876. Incorporation of formate-C'‘ into
nucleic acids of Ehrlich ascites tumor cells.
HeLtEN HarrineTton* anp Pau §. Lavix.
Western Reserve Univ. Atomic Energy Project,
Cleveland, Ohio.

The incorporation of formate-C!‘ by tumor cells
of mice inoculated 6-7 days previously has been
studied in vitro and, in vivo. In the in vitro ex-
periments the tumor cells were removed, washed,
suspended in saline and incubated with formate-
C for 3 hr. at 37°C. In the in vivo experiments
mice were injected intraperitoneally with for-
mate-C!* and killed 3 hr. later. Nucleic acids were
extracted from the cells with hot 10% NaCl,
precipitated with ethanol, separated by alkaline
incubation and hydrolyzed. Specific activities of
purines and pyrimidines were determined after
separation and purification by ion exchange and
paper chromatography. After incubation of the
tumor cells with formate-C'4 in vitro, the specific
activity of DNA thymine was 30-40 times that of
the DNA purines. In the in vivo experiments, the
specific activity of DNA thymine was similar to
that of DNA purines. It is possible that the in
vitro system is deficient in purine synthesis, or
that in the in vitro system, formate-C' enters
DNA thymine by a fairly direct pathway in which
there is less opportunity for dilution and diversion
of the isotope than in DNA purine synthesis.
x-Irradiation of the cells in vitro with 10,000 r
did not affect the in vitro incorporation of for-
mate-C!4 into RNA purines but inhibited its
incorporation into DNA purines and thymine by
30-40%. Irradiation of the tumor-bearing mice
with 1000 r appeared to affect the in vivo incorpora-
tion of formate-C'4 into DNA guanine more than
into other nucleic acid bases.

877. Stereospecificity of mushroom tyrosin-
ase. JoSEPH Harris AND DanrEL J. CAVEN-
auGuH (introduced by Joun A. Muntz). Dept. of
Biochem., Albany Med. College, Albany, N. Y., and
Dept. of Pharmacology, Univ. of Tenn. School of
Medicine, Memphis.

The oxidation of .L-tyrosine by mammalian
tyrosinase is known to be specific; p-tyrosine is

not oxidized (A. B. LERNER, Adv. in Enzymol. 14:

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS 269

81, 1953). A similar specificity, hitherto unrecog-
nized, has been observed for mushroom tyrosinase.
This specificity did not extend to t-DOPA and
p-DOPA. An effect attributable to asymmetry was
also found with the optical antipodes of leucyl-.-
tyrosine peptides. p-leucyl-L-tyrosine was re-
fractory to oxidation, while .L-leucyl-L-tyrosine
was oxidized readily. The order of reactivity of
selected tyrosine dipeptides was found to be:
L-tyrosine > glycyl-L-tyrosine > N-chloracetyl-
L-tyrosine = L-leucyl-L-tyrosine > >>> p-leucyl
-L-tyrosine. The relative susceptibility of tyrosine
dipeptides to tyrosinase action could not be corre-
lated with their ability to complex Cu**. By means
of an ascorbic acid-Fe hydroxylation procedure
(UDENFRIEND, CLARK, AXELROD AND BRopIzE,
J. Biol. Chem. 208: 731, 1954), the corresponding
‘dihydroxyphenylalanine’ analogs of the peptides
were prepared and then subjected to the action of
tyrosinase. Introduction of the second hydroxyl
group abolished stereospecificity. The refractory
p-leucyl-L-tyrosine, when converted to the ‘dopa’
analog, was oxidized. Similarly, the rates of
oxidation of the tyrosine dipeptides after hy-
droxylation were increased and the induction
period characteristic of monophenolic substrates
eliminated. Whereas the results of previous in-
vestigations underline a close relationship be-
tween the ‘cresolase’ and ‘catecholase’ activities
of tyrosinase, the present work reveals a more
rigid stereochemical requirement for cresolase
activity. The catalytic sites associated with the
2 types of activity appear to differ in spatial
orientation.

878. Phosphorolysis of glycinamide ribotide.
StanpisH C. Hartman (introduced by L. G.
Wesson). Div. of Biochemistry, Massachusetts
Inst. of Technology, Cambridge.

The investigation of the enzymatic formation
of glycinamide ribotide (GAR), an early inter-
mediate in the de novo synthesis of inosinic acid,
has been continued with fractionated extracts of
chicken liver. The synthesis of GAR from glycine,
glutamine, ATP and phosphoribosyl pyrophos-
phate (PRPP) requires 2 enzyme fractions.
Enzyme I has been purified approximately 100-
fold from chicken liver extract and enzyme II
about 30-fold. Enzyme I catalyzes the formation
of glutamic acid from glutamine and PRPP. In
the presence of ADP and orthophosphate, enzyme
II effects the release of glycine from GAR, a re-
versal of the synthetic reaction. Arsenate can re-
place the requirement for phosphate. However,
neither AMP nor ATP can substitute for ADP.
A reaction between P*-labeled orthophosphate
and ADP to form labeled ATP has been shown to
have an absolute requirement for GAR. This
synthesis of a pyrophosphate linkage of ATP

270

results from the phosphorolysis of an amide bond.
Since ADP is required for P*-phosphate in-
corporation into ATP, and 0.8 m of ATP is synthe-
sized per mole of glycine formed from GAR, a
direct synthesis of ATP, and not an exchange
reaction between the terminal phosphate group of
ATP and inorganic phosphate, is suggested.
Glycine will not catalyze the turnover of P** into
ATP. These data tend to substantiate the pro-
posal of Goldthwait et al., (Biochim. et biophys.
acta, 18: 148, 1955), that 5-phosphoribosylamine
is the carbohydrate intermediary in these reac-
tions. Attempts to isolate such a compound from
an enzymatic system have so far proved unsuc-
cessful.

879. Hexose oxidation by heart muscle. NIELS
HavuGaaRD AND Haro.tp Itsxovitz.* Lab. of
Pharmacology, Univ. of Pennsylvania School of
Medicine, Philadelphia.

Tissue homogenates fortified with KCl and DPN
have been shown to oxidize glucose completely
(WENNER, DunN AND WEINHOUSE, J. Biol. Chem.
205: 409, 1953). We have studied the properties of
this multi-enzyme system in rat heart homo-
genates with the following results: 1) glucose,
fructose and mannose are oxidized at equal rates,
galactose less rapidly. 2) Under optimal conditions
intermediates do not accumulate, 1 um of glucose
utilized giving rise to 6 um of carbon dioxide and
using 6 uM of oxygen. 3) By centrifugation inac-
tive soluble and particulate fractions are obtained.
Activity is restored by combining these fractions.
4) Activity is dependent on the ionic environ-
ment. Sodium ions are inhibitory. Potassium and
magnesium ions singly or together activate the
system. The effects of Kt and Mg** are functions
of the ratios Kt/Na* and Mg*t*/Nat rather than
of the absolute concentrations. Calcium is in-
hibitory. 5) SH-groups are necessary for activity.
Inhibition is caused by p-chloromercurobenzoic
acid an@ 1 atm. oxygen, especially in the presence
of traces of copper. It appears that the soluble
glycolytic enzymes and the particulate enzymes
of the tricarboxylic acid cycle function in such an
organized way that they constitute a unit with
distinct properties. With such a preparation the
physiological regulation of enzyme reactions may
be studied in a noncellular system. This approach
is exemplified by our studies of ionic effects.

880. Metabolism of internally labeled S*-
proteins. Frrrx Havurowitz aNnp Harry
WaxTeR.* Dept. of Chemistry, Indiana Univ.,
Bloomington.

Rats were intravenously injected with S**-amino
acids or with rabbit or guinea-pig serum albumin
containing S*-amino acids. In some experiments
the S*-proteins were coupled with traces of I'

FEDERATION PROCEEDINGS

Volume ij

or of diazotized C'‘-anthranilic acid. The live
proteins, 9 days after injection, contained 24%
of the injected S* and about 1% of the injected
C4, but only about 0.02% of the injected [i
When rabbits were injected with the doubly
labeled rabbit or guinea-pig protein, the ratio
§$%/T)3! or §%/C!4 in their serum proteins wag
close to that of the injected protein, but wag
higher in lung, spleen and bone marrow protein;
it was highest in the liver proteins in agreement
with our previous work (FRIEDBERG et al., J. Im.
munol. 75: 315, 1955). These results indicate that
the injected protein remains unchanged only in
the blood plasma and that the excess of S** over
[!3! in the tissues is caused by the loss of I!*! rather
than by reutilization of fragments containing
S*. After injection of S*-amino acids the highest
concentration of protein-bound S* was found in
the cytoplasmic granules of the liver homogenates,
However, when S*-proteins were injected the
protein-bound radioactivity was first evenly
distributed among all of the subcellular fractions
and after several weeks was highest in the nuclear
fraction. Evidently, the metabolism of injected
$*-proteins is different from that of free S*-amino
acids. The former seem to be incorporated into
the tissue protein fractions as such or, by trans-
peptidation, as large fragments without previous
breakdown to amino acids. (Supported by The
Natl. Science Fndn., the Public Health Service,
the Atomic Energy Commission and the Office of
Naval Research.)

881. Effect of ACTH on the corticosteroid
content of adrenal tissue. Rosert C.
Haynes, Jr. AND Lucite BerTHET (introduced
by Jack Lronarps). Dept. of Pharmacology,
Western Reserve Univ., Cleveland, Ohio.
Experiments were performed to determine

whether ACTH acts on the adrenal cortex by: 1)

primarily increasing the rate of steroid synthesis

with a resultant ‘spilling’ of these corticosteroids
into the extracellular fluid, or 2) primarily causing
release of the steroid hormones from the adrenal
cells with the resultant mass action effects causing
an increased rate of synthesis. Tissue slices from
beef adrenal cortices were incubated in Ringer’s
solution for 20 min. (after a 45-min. preincubation)
with and without added ACTH. They were then

washed and added to cold acetate buffer (px 3.5)

and homogenized in this buffer with added 10%

sucrose. The slice corticosteroids were then puri-

fied and assayed by conventional techniques. It
was found that slices stimulated by ACTH hada
corticosteroid content which was greater than
that of the control slices, indicating the primary
effect of ACTH is on synthetic mechanisms, for
had the primary action of ACTH been on corti-
costeroid release, the intracellular content should

olume i

‘he liver
ed 2-4%
injected
ted Ji,

doubly
he ratio
ins was
but wag
protein;
reement
J. Im
ate that
only in
S*5 over
1 rather
taining
highest
ound in
renates,
ted the
evenly
ractions
nuclear
njected
5-amino
ed into
y trans-
Trevious
by The
Service,
)ffice of

steroid
RT ¢,
oduced
icology,

ermine
by: I)
nthesis
teroids
sausing
idrenal
-ausing
8 from
inger’s
vation)
e then
oH 3.5)
d 10%
1 puri-
ues. It
hada
- than
rimary
as, for

corti-
should

March 1956

have been diminished. Most of the ACTH-induced
increase in slice corticosteroid content was found
in the soluble portion of the cells. This was deter-
mined by centrifuging homogenates of the slices
and determining the corticoid content of the
sedimented and supernatant fractions. Because
these experiments indicated ACTH acts primarily
on synthesis rather than release, studies are now
being carried on to learn more about the manner
in which ACTH affects these synthetic processes.

882. Biosynthesis of deoxyribonucleic acid in
slices of regenerating rat liver. LIsELoTTE I.
Hecut* anp Van R. Porter. McArdle Lab.,
Univ. of Wisconsin, Madison.

The rate of deoxyribonucleic acid (DNA)
labeling from orotic acid-6-C* in slices of 3, 11,
15, 19, 24 and 48 hr. regenerating livers during a
4-hr. period was examined. No DNA labeling was
observed in the slices of 3-15 hr. regenerating
livers, wide biological variations were obtained
19 hr. postoperative and a high rate of DNA label-
ing occurred 24 hr. postoperative decreasing to a
lower rate in 48 hr. regenerating liver in agree-
ment with previous in vivo studies. A comparison
of the extent of DNA labeling in vivo during the
17- to 19-hr. period postoperative with the rate of
DNA synthesis in slices of the same livers indi-
cated a close parallelism between the in vivo and
in vitro rate of DNA labeling. The production of
the DNA synthetic mechanism could not be
demonstrated in the tissue slices. Using orotic
acid-6-C!4 as a precursor for the labeling of DNA
in vitro the DNA thymine had a higher specific
activity than the DNA cytosine at early time
points. Totally labeled C'* cytidine was incor-
porated more extensively into DNA deoxy-
cytidylic acid than thymidylic acid and labeled
both the base and deoxyribose moieties. The
extent of the utilization of orotic acid and the
nucleosides as precursors for the synthesis of
nucleic acids in the slice was of the same magni-
tude, and competition between the precursors was
observed affording an opportunity to study the
metabolic sequences in DNA biosynthesis.

883. Metabolism of a- C!4-DL-hydroxyproline
in the intact rat. Radioactivity in amino
acids. WatteR W. Heck, Joun C. Leak,
GzrorcE WoLF aND CLEMENS R. A. BERGER
(introduced by H. H. Mrtcuety). Radiocarbon
Iab. and Div. of Animal Nutrition, Univ. of
Illinois, Urbana.

Radioactive a-C!4-pL-hydroxyproline was pre-
pared by condensation of 3-phthalimido-l1 ,2-
epoxypropane with diethyl sodiomalonate-2-C'4,
chlorination, hydrolysis of the chlorolactone so
obtained, cyclisation to hydroxyproline-C' and
separation into the natural and the allo isomers
through crystallisation of the copper salts (overall

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

271

yield, 17.1%). The radioactive natural and allo-
puL-hydroxyproline (15 mg of each, 664 uc/mmM)
were each separately injected intraperitoneally
into rats. The expired carbon dioxide contained
25.1% (natural) and 11.8% (allo) of the activity.
With the natural isomer, the principal metabolite
in liver protein was found to be alanine, with the
highest activity and specific activity, followed by
aspartic and glutamic acids, in that order. Gly-
cine, serine, methionine and proline contained low
levels of activity. Radioactive hydroxyproline
could be isolated from purified liver protein. A
very small percentage of the activity of the liver
nonprotein fraction was contained in y-hydroxy-
glutamic acid. No radioactive y-hydroxyornithine
could be detected in liver or urine. The principal
urinary metabolite was found to be a strongly
basic compound. The metabolic pattern of allo-
pu-hydroxyproline was closely similar to that of
the natural isomer. These results suggest 2 main
metabolic pathways for hydroxyproline, after
opening of the ring to hydroxyglutamic acid or a
closely related compound: breakdown to alanine
and a 2-carhon fragment such as glyoxylic acid; or
oxidative decarboxylation to aspartic acid.

884. Metabolism of uracil in normal and neo-
plastic tissues. CHARLES HEIDELBERGER AND
EseruarD Harsers.* McArdle Memorial Lab.,
The Med. School, Univ. of Wisconsin, Madison.
We have previously reported (Federation Proc.

14: 248, 1955) the considerably increased in vivo
utilization of uracil-2-C4 for nucleic acid pyrimi-
dine biosynthesis in the Flexner-Jobling carci-
noma and intestinal mucosa relative to normal
liver. Since the radioactivities in the nucleic acids
paralleled those of the acid-soluble uridine nucleo-
tides, it is believed that the differences among the
tissues reflect their ability to convert uracil into
nucleic acid precursors. Accordingly the conver-
sions have been studied in vitro in the soluble,
high speed supernatant system developed by
Hurlbert and Reichard (Acta Chem. Scand. 9: 251,
1955). Uracil-2-C'* and orotic acid-6-C'* were
incubated in separate aliquots of the soluble
fraction obtained from homogenates of normal
liver, intestinal mucosa and Flexner-Jobling
carcinoma. No C!4O, was evolved, and the acid-
soluble extracts were chromatographed on
Dowex-1 with extended gradient elution. In the
liver and tumor systems, orotic acid was con-
verted almost completely into the various uridine
nucleotides, with UTP predominating; in the
intestinal preparation some orotic acid was re-
covered unchanged. In all 3 tissue systems uracil
was recovered unchanged, showing that, unlike
orotic acid, uracil is not converted into nucleo-
tides in the supernatant fraction under the condi-
tions used.

272

885. Cross reactions of glycogen and limit
dextrin in antipneumococcus sera. MICHAEL
HEIDELBERGER, BERTIL BJORKLUND,* FELIX
CorpoBa* anp Hans JAHRMARKER.* College of
Physicians and Surgeons, Columbia Univ., and
Inst. of Microbiology, Rutgers Univ., New Bruns-
wick, N. J.

Following the demonstration (J. Exper. Med.
99: 343, 1954) that glycogen displays similar
immunological reactivity to that of other poly-
glucoses such as dextran, a quantitative com-
parison was made of the precipitation of several
antipneumococcus horse and/or rabbit antisera of
types II, IX, XII, XX and XXII by the principal
fraction of oyster glycogen and the limit dextrin
prepared from it by the action of B-amylase. In
all except antisera to type IX pneumococcus, the
limit dextrin precipitated more antibody than did
the glycogen. This indicates that, except in this
one instance, increased accessibility of the a-1,4,6-
branch points of glucose to the antibody increases
the extent of the cross reaction. The data do not
suffice alone to determine whether cross-reactivity
is due to the multiple branch points or solely to
multiple a-1,6-linked units of glucose. In the
relatively fresh rabbit antisera, especially, the
action of a-amylase on the specific precipitates
was often quite rapid but could be at least par-
tially corrected for.

886. Substances containing sialic acid. RALPH
HeIMER* AND Karu Meyer. Dept. of Medicine,
Columbia Univ., College of Physicians and
Surgeons, New York City.

A number of mucoids containing ‘sialic acid’
have been studied. The most thoroughly investi-
gated of these, prepared from bovine submaxillary
gland, appeared homogeneous on electrophoresis
and ultracentrifugation, and contained 9.1%
nitrogen, 19% sialic acid, 17.5% hexosamine, and
no other detectable hexoses. It strongly inhibited
influenza virus hemagglutination, but this in-
hibiting power was lost after incubation with
extracts of pneumococcus and Vibrio cholerae.
After this treatment, 30%-50% of the ‘sialic acid’
and small amounts of hexosamine fragments
became dialyzable, but there was no release of free
amino groups. Concomitantly there was a fall in
viscosity, and paper chromatography revealed
two ‘sialic acid’ spots. Whether hydrolysis was
performed by enzymes or mild acid, the crystalline
compounds obtained possessed similar properties.
‘Sialic acid’ of cow colostrum was obtained in
crystalline form after acid hydrolysis of non-
dialyzable material. Its methoxy derivative,
lactaminic acid, has been previously prepared
(Kuhn). Infrared spectrum of this laboratory’s
preparation exhibits marked similarity with
preparations obtained from submaxillary gland.

FEDERATION PROCEEDINGS

Volume i§

Dialyzable portions of cow colostrum contained an
oligosaccharide composed of ‘sialic acid’ and lage.
tose. A preparation of Lactobacillus bifidus, var,
Penn., cleaved oligosaccharide’s beta-galactosidie
linkage, and gave paper chromatographic evidence
of a ‘sialic acid’ linked hexose. (Supported by a
grant (A-570) from the Public Health Service.)

887. Relationship of chloride transport to
gastric acid secretion. Reich HEINZ anp
Ricuarp P. Dursin.* Biophysical Lab. Harvard
Med. School, and Dept. of Biochemistry, Tufts
Med. School, Boston, Mass.

In experiments on the frog gastric mucosa in
vitro, H* secretion and chloride fluxes in both
directions have been measured under various
conditions of metabolic and secretory activity,
Decreasing the chloride concentration in the
secretory solution by a factor of 5 markedly
decreases the chloride flux from the nutrient to
the secretory side. The addition of 2:4 dinitro-
phenol to the nutrient solution decreases the
membrane conductivity and the chloride fluxes in
each direction by a factor of 2-3. The chloride
fluxes in both directions increase with increasing
H* secretion. The above findings are interpreted
in terms of a metabolically linked carrier transport
of chloride across the mucosa. It is also found
that the gastric potential can be abolished com-
pletely by addition of Diamox followed by hista-
mine while acid secretion continues at a normal
rate.

888. Enzymatic conversion of pteroylglutamic
acid to citrovorum factor by partially
purified extracts of Lactobacillus casei.
C. R. Hets_er* anp B. 8. ScHWEIGERT. Ameri-
can Meat Inst. Fndn. and Dept. of Biochemistry,
Univ. of Chicago, Chicago, Ill.

An enzyme system that actively converts
pteroylglutamic acid (PGA) to the citrovorum
factor (CF) has been fractionated and partially
purified from sonic extracts of Lactobacillus casei.
The test system included phosphate buffer, ATP,
DPN, Mg ions, serine and ascorbic acid. CF was
assayed microbiologically using Pediococcus
cerevisiae 8081 as previously reported (Federation
Proc. 14: 436, 1955). The supernatant from soni-
cated L. casei cells was centrifuged at 25,000 X 9,
and fractionated with ammonium sulfate. The
fraction precipitated by 50-60% of saturation
gave a 15% conversion of PGA to CF, but the
conversion was doubled when the precipitate from
60-75% of saturation (inactive itself) was added.
This suggested that more than one enzyme was
involved in this conversion or that a cofactor was
being split off at the higher concentrations. Boiled
supernatant preparations approximately doubled
this conversion when added to dialyzed and non-

Ss se © so mH SS sh =

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lume §

ined an
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IS, Var,
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ridence
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ort to
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Larvard
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08a in
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ent to
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A meri-
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ATP,
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. The
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March 1956

dialyzed ammonium sulfate fractions, as well as
when added to the untreated supernatant. Similar
increases in the total conversion were also noted
upon the addition of boiled saline extracts of
acetone powder, or concentrated dialysates of un-
fractionated supernatant preparations. Increased
levels of ATP, DPN, Mg ions, serine and ascorbic
acid failed to enhance the conversion. Experiments
designed to further elucidate this enzymatic
conversion of PGA to CF are being continued.

889. Use of rabbit aortic strips to biologically
differentiate two forms of angiotonin. O. M.
Heimer. Lilly Lab. for Clin. Research, General
Hosp., Indianapolis, Ind.

Previously it has been reported (Federation
Proc. 14: 225, 1955) that a factor is present in
plasma which enhances the contraction produced
by angiotonin on spirally cut rabbit aortic strips.
The factor has characteristics of an enzyme. An
amount of angiotonin which will not cause con-
traction of the strip will, after incubation with
the factor, induce strong contraction. Before and
after activation, however, angiotonin has the
same pressor activity when injected intravenously
in a pithed cat. Since Skeggs et al. (J. Exper.
Med. 99: 275, 1954) by means of countercurrent
distribution technique has demonstrated 2 forms
of angiotonin (Hypertensin I and Hypertensin IT)
a sample of angiotonin which had been incubated
with human plasma was: subjected to counter-
current distribution. Although the sample proved
to be a mixture, that portion which on the curve
corresponded to Hypertensin I was inactive on the
strip, whereas the fraction corresponding to
Hypertensin II caused a strong contraction. Both
showed the same pressor activity when injected
intravenously in cats. These findings were con-
firmed with highly purified Hypertensin I and II
and purified converting enzyme kindly supplied by
Skeggs. The rabbit aortic preparation offers a
biological means of differentiating these 2 forms
of angiotonin. Some hypertensive patients have a
higher content of the converting enzyme than
normotensive subjects.

890. Metabolism of C!*-tryptophan in the rat.
L. M. Henperson anp L. V. Hanxss. Dept.
Chemistry and Chemical Engineering, Univ. of
Illinois, Urbana, and Med. Dept., Brookhaven
Natl. Lab., Upton, N. Y.

Tryptophan labeled in positions 7, 7a, and 3a of
the benzene ring (numbering system, Elderfeld,
Heterocyclic Compounds, vol. 3, p. 6) and tryp-
tophan labeled in the alpha-carbon atom was
administered to adult male rats at dosages of
11-51 mg. Exereta, expired carbon dioxide, and
tissues were collected for analysis for C!4 content.
Approximately } of the C' from ring-labeled

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

273

tryptophan appeared as expired CO:; 12-17%
appeared in the urine and a small percentage in
feces. Sixteen per cent was found in the animals’
tissues 24 hr. after a single dose; 30% after 4 doses
distributed over 48 hr. of administration. The
quinolinic acid isolated from the urine had specific
activity equal to approximately 70% of that
expected if the quinolinic acid were derived solely
from the administered ring-labeled tryptophan.
The carboxyl group in the 2-position of the
quinolinic acid contained the expected 25% of the
isotope, suggesting that this atom arises from the
7-position of the tryptophan. The N’methyl-
nicotinamide isolated from the urine had 20-30%
of the C' activity that it would have had if
derived solely from the administered ring-labeled
tryptophan. When alpha-C'!-tryptophan was
given 35-50% of the C'4 was expired as C1O2.
More than half of the C'4 excreted in the urine
following the administration of both types of
labeled tryptophan was present in compounds
other than urea and identified tryptophan ca-
tabolites. (Supported by the Atomic Energy
Commission.)

891. Chemical and enzymatic studies of poly-
nucleotides synthesized by polynucleotide
phosphorylase. Lron A. HeEppret, J. D.
SmituH,* Priscitua J. Ortiz,* anp SEVERO
Ocuoa. National Institutes of Health, Bethesda,
Md., and Dept. of Biochemistry, New York Univ.,
College of Medicine, New York City.

Studies of the highly polymerized ribopoly-
nucleotides synthesized by polynucleotide phos-
phorylase (M. Grunsperc-Manago, P. J. Ortiz
AND S. Ocuoa, Science, 122: 907, 1955) show that
they possess the structural pattern of natural
RNA. Ribonuclease digestion of the uridylic acid
(UMP) polynucleotide yields first high concentra-
tions of the cyclic dinucleotide, and later cyclic
uridylic acid (uridine-2’:3’ monophosphate) and
3’-UMP. Digestion of the adenylic-uridylic (A-U)
and the adenylic-guanylic-uridylic-cytidylic (A-
G-U-C) polynucleotides yields pyrimidine mono-
nucleotides and small polynucleotides with
3’-phosphomonoester end-groups. Eight of these
polynucleotides have been characterized and are
the same as obtained from RNA. The occurrence
of links between adenylic acid and the other
mononucleotide units in A-G-U-C will also be
illustrated by experiments with a polynucleotide
containing P*-labeled adenylic acid. End-group
assays (R. Marxuam, R. E. F. Matruews, AND
J.D. Smrtu, Nature, 173: 5387, 1954) of some of the
biosynthetic polymers have shown that the chains
vary in length from 30 to 250 mononucleotide
units and are terminated by 5’-phosphomonoester
groups. Nucleoside-5’-monophosphates as such
appear not to act as primers.

274

892. Specificity of myosin-water interaction
in hydrolysis of adenosinetriphosphate.
Ear B. Herr, Jr.* AND DANIEL E. KosHLanp,
JR. Biology Dept., Brookhaven Natl. Lab., Upton,
We f..

The role of water in reactions of hydrolytic
enzymes remains obscure because of its high
concentration and the difficulty of varying its
concentration significantly without changing the
properties of the solvent. To clarify the water-
protein interaction a procedure has been devised
which depends on a) the relative reactivities of
water and methanol in nonenzymic reactions on
analogous compounds and b) the detection of
minute amounts of the methyl compound by
isotopic methods. Myosin was chosen because it
has been shown that the enzyme has no gross
transferase activity (Morton, Nature 172: 65, 1953)
and that it catalyzes an attack on the phosphorus
atom (KosHLAND, BUDENSTEIN AND KoWALSKY.
J. Biol. Chem. 211: 279, 1954). Studies of acid-
catalyzed displacements on phosphorus with
compounds such as creatine phosphate and sodium
pyrophosphate showed that the relative re-
activities of water and methanol were approxi-
mately equal. Adenosine-triphosphate was then
hydrolyzed in the presence of purified myosin and
10-4 m C!4H;OH. The amount of methyl phosphate
formed by the enzyme action was determined by
addition of inactive carrier and determination of
the specific activity of the subsequently separated
and purified methyl phosphate. No detectable
amounts of radioactivity were found, showing
that the water was at least 80 times more reactive
than methanol in the myosin reaction. This is in
conjunction with the nonenzymic studies, demon-
strates that the water does not act directly from
solution, that there is a specific myosin-water
interaction and that this interaction is sufficiently
specific to exclude the closest homologue of water,
i.e. methanol. (Supported by the Atomic Energy
Commission.)

893. Effect of estradiol on metabolism of
serine-3-C"‘ in surviving uterine segments.
AILENE HERRANEN* AND GERALD C. MUELLER.
McArdle Memorial Lab., Univ. of Wisconsin,
Madison.

Previous studies on the metabolism of formate
and glycine-2-C!‘ in surviving uterine segments
from estrogen-treated rats have revealed the ‘one
carbon’ metabolic pathways as highly sensitive
indicators of early estrogen action. These studies
have been extended with serine-3-C‘. In response
to 6 hr. pretreatment with estradiol, the incorpora-
tion of radioactivity into the purine bases of mixed
nucleic acids was accelerated 5-6-fold. The in-
corporation into protein and oxidation to carbon
dioxide was accelerated 2-fold; no effect was

FEDERATION PROCEEDINGS

Volume 1§

observed in the lipide fraction. The addition of
nonradioactive formate to the reaction flask acted
as a trap for the one carbon unit derived from the
serine-3-C'4 thereby obscuring the effect of estrogen
action on the one carbon pathway into purines
and carbon dioxide. Determination of the amount
of radioactivity trapped by the pool of non-
radioactive formate demonstrated that the
estrogen pretreatment had accelerated the meta-
bolic reactions yielding formate from serine-3-C1,
The data support the concept that estrogen pre-
treatment accelerates independently a number of
steps in the metabolism of one carbon compounds;
these steps largely concern reactions involving
carboxyl groups.

894. Aminobutyric acid as a_ pyrimidine
precursor in Neurospora. RoBErt L. HErr-
MANN* AND JAMES L. Farriey. Kedzie Chemical
Lab., Michigan State Univ., East Lansing.

The ability of alpha-aminobutyric acid to
support the growth of certain pyrimidine-re-
quiring mutants of Neurospora crassa has been
reported previously (FarrRLEY. J. Biol. Chem. 210:
347, 1954). We have now carried out experiments
designed to determine if the growth-supporting
action of this compound is related to its use in
this organism as a precursor of the pyrimidine
ring. The mold was allowed to grow in a medium
containing aminobutyric acid-3-C, the nucleic
acid pyrimidines were isolated and their content
of radioactive carbon was measured. In a typical
experiment using mutant strain 1298 and amino-
butyric acid with a specific activity of 4.8 X 104
cpm/uM, each of the pyrimidines, uracil and
cytosine, was found to have an activity near
5.3 X 10? cpm/uM, a dilution of the isotope by a
factor of 9. A 30-fold dilution of the isotope would
have been obtained had the aminobutyric acid
served equally as well as a general carbon source
as the other carbon-containing compounds of the
medium, sucrose and tartrate. For comparison,
the nucleic acid purines, adenine and guanine,
were labeled to the extent of about 1 X 103 cpm/uM,
a dilution factor of 48. In similar experiments with
a wild-type strain of the mold, dilution factors of
30 for the pyrimidines and 80 for the purines
resulted. (Supported by a contract with the
Atomic Energy Commission.)

895. Competitive role of tungsten in molyb-
denum nutrition. Epwin 8. Hiearns,* Dan A.
RicHERT AND W. W. WEsTERFELD. Dept. of
Biochemistry, State Univ. of New York, College of
Medicine, Syracuse.

Chicks fed a synthetic low-Mo ration containing
either 4.5 or 9.4mg% W (as Na2WQ,) developed an
apparent Mo deficiency. Growth rates were
depressed 8 and 19%, respectively, in 5 wk.;

lume 16

tion of
k acted
om the
trogen
yurines
mount
f non-
t the
meta-
-3-Cu,
N pre-
iber of
ounds;
olving

1idine
HERR-
emical

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n. 210:
ments
orting
use in
nidine
edium
ucleic
mntent
ypical
mino-
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March 1956

mortality was 24 and 28%. Liver and other tissue
Mo concentrations were less than 10% of the
normal values, while the xanthine dehydrogenase
activities of small intestine, liver, kidney and
pancreas were reduced to less than 15% of normal
by both diets. Uric acid excretion was decreased
50%; the missing uric acid was replaced by an
equivalent amount of a mixture of xanthine and
hypoxanthine. All of these effects were completely
reversed, and normal values were restored, by
adding 2 and 6 mg% Mo (as Na:MoQ,) respectively
to the diets containing tungstate; except that
with the 9.4 mg% W-diet, growth was still de-
pressed 4% and mortality was 9%. Weanling rats
fed similar W-containing diets had essentially no
xanthine oxidase activity (as measured mano-
metrically) in the small intestine, liver, kidney,
lung or spleen. Tissue Mo concentrations were
reduced to 10 and 5% of normal values by the 4.5
and 9.4 mg% W-diets, respectively. These tissue
effects were completely reversed by supplementing
the W-diets with 2 and 6 mg% Mo. Other than the
loss of tissue Mo and xanthine oxidase, no un-
toward effects were observed in W-fed rats. They
grew normally and continued to excrete uric acid
as the only major urinary purine. A 75-mg dose of
xanthine was oxidized to uric acid in a normal
fashion by such tungstate-fed rats.

896. Metabolism of DL-glutamic acid-2-C'* by
the rat: apparent conversion in vivo of
citrate to oxalacetate and acetate. ROBERT
J. Hitt AND Rocer E. Koepre (introduced by
T. P. Nasu, Jr.). Div. of Chemistry, Univ. of
Tennessee, Memphis.

Although the enzymatic reactions necessary to
convert a-ketoglutarate to oxalacetate and acetate
(via citrate) have been shown to be reversible
in vitro, there appears to be no evidence concerning
such a reversal in vivo. In attempting to demon-
strate such a reversal in vivo we have administered
pL-glutamic acid-2-C!4 to rats by intraperitoneal
injection. The animals were killed after 4 hr. and
aspartic and glutamic acids isolated from liver
and carcass proteins. The isolated aspartic acids
were found to have 30-60% of their carbon 14
located in the noncarboxy] portion of the molecule.
All carbons of the isolated glutamic acids con-
tained radioactivity. Conversion of glutamate-2-
C4 to a-ketoglutarate and hence to succinate
should yield aspartate labeled only in the carboxyl
carbons and glutamate labeled primarily in
carbons 1 and 2. Equilibration of a-ketoglutarate
with the tricarboxylic acids prior to its conversion
to succinate would not alter the predicted labeling
patterns. Data have been accumulated which
clearly demonstrate a pathway(s) of glutamate
catabolism other than a direct conversion to
succinate via a-ketoglutarate. These data appear

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

275

to be compatible with a conversion of a-keto-
glutarate to oxalacetate and acetate, via citrate.
(Supported by the Natl., Science Fndn.)

897. Succinoxidase system of Myrothecium
verrucaria. JAMES L. HItTon* AND FREDERICK
G. Sir. Dept. of Botany, Iowa State College,
Ames.

Mycelial pellets grown in liquid shake-culture
were ground with powdered glass in a glass
homogenizer in pH 7 0.01 m phosphate. The particle
fraction sedimented at 20,000 X g contained all the
succinoxidase activity. Most preparations were
stimulated by added cytochrome c, and this was
increased by washing. With added cytochrome c
and glutamate the optimum pH was about 7.2,
the Kn 0.007 m, and the average Qo, (N) 580.
Succinoxidase activity was consistently stimu-
lated by ADP, ATP, and UTP, in some cases by
AMP, but in no case by UMP. Stimulation by
0.001 m ATP ranged from 50 to 100% and was
inhibited by versene. This and other evidence
indicated that cations including Mg*tt were
probably involved. Succinate oxidation was
inhibited by malate, fumarate, oxalacetate, Cott,
Cu**, cyanide, malonate and the alkylnaphtho-
quinone, SN5949, and ATP reversed inhibition by
the first four. ATP stimulation appeared to
involve the removal of oxalacetate formed by
malate oxidation too slow to detect mano-
metrically. Furthermore, glutamate was even
more effective than ATP in stimulating succinate
oxidation and in reversing inhibition by added
oxalacetate, apparently by transamination of the
latter to aspartate. Reciprocal plots (LINEWEAVER
AND Burk) showed that ATP and glutamate did
not change the Vmax but appeared to remove a
competitive inhibitor. Finally, comparison of
sensitivity of mycelial respiration and particle
succinoxidase activity to malonate, cyanide, Cot*
and SN5949 indicated an essential role for the
enzyme system in respiration. (Supported in part
by a contract with the Office of Naval Research.)

898. Glycogen metabolism of Tetrahymena
pyriformis. James F. Hoge ann ConrapD
WaGneErR (introduced by Lawrence Lovts).
Dept. of Biological Chemistry, Univ. of Michigan,
Ann Arbor.

Washed cell suspensions of Tetrahymena pyri-
formis E show a doubling of glycogen content
under endogenous conditions. This synthesis of
glycogen occurs only under aerobic conditions
whereas fermentation of the glycogen takes place
anaerobically. The endogenous synthesis is de-
pendent on the age of the cultures as well as the
initial cellular levels of glycogen and phospholipid.
Concurrent with the glycogen synthesis there are
large decreases of cellular lipids and of protein

276

nitrogen. The decrease in lipid is primarily of
phospholipid while the lost protein N appears as
nonprotein N, mainly ammonia N. The decrease
of either lipid or protein alone is adequate to
account for the observed synthesis of glycogen.
When the cells are incubated in the presence of
glucose, the increase of glycogen synthesis above
endogenous levels accounts for all of the added
glucose. Neither single amino acids nor a mixture
of the 11 essential amino acids stimulated glycogen
synthesis, although ammonia production was
stimulated. The addition of butyrate, acetoacetate
or glycerol oleate, however, produced a large
stimulation of glycogen synthesis. Pyruvate and
glycerol were without effect. A determination of
the endogenous respiratory quotient showed a
value of 0.63 which is compatible with a conversion
of either protein or lipid to carbohydrate. In the
presence of glucose, the respiratory quotient
decreased to 0.48.

899. Effects of cholinergic agents on in-
corporation of P® into phospholipids of
cell-free preparations of brain cortex.
Lowe. E. Hoxkin anp IRENA MazurRKIEWICz.*
Dept. of Pharmacology, McGill Univ., Montreal,
Canada.

Earlier studies showed that acetylcholine or
carbamylcholine stimulated the incorporation of
P% into the phospholipids of slices of pigeon
pancreas and guinea pig brain cortex. This stimu-
lation was shown to be due to an increased turn-
over of phosphate in preformed phospholipid and
to take place mainly in the inositol-containing
phospholipids. These studies have now been
extended to cell-free preparations. Homogenates
or washed preparations of mitochondria plus
microsomes prepared in isotonic sucrose were
incubated for 1 hr. at 37° with 0.10 m glycy!l-
glycine, pH 6.6, 0.001 m adenylic acid, 0.001 m
sodium fumarate, 0.02 m sodium pyruvate, 2 X 10-°
M cytoehrome c, 0.007 M magnesium sulfate and
0.001 m disodium monohydrogen phosphate (all
concentrations are final). P®* was incorporated
quite rapidly into the phospholipids of brain
cortex homogenates but much more slowly into
the phospholipids of pancreas homogenates; 10-° m
acetylcholine or 10-° m carbamyicholine usually
increased the incorporation of P* into the phos-
pholipids of brain cortex homogenates about 25 to
50%. This stimulation is less than was previously
observed in slices, but only one-thousandth the
concentration of cholinergic agent is required to
achieve the maximal effect. No stimulation by
cholinergic agents was observed in pancreas
homogenates. The phospholipids of a washed
mitochondrial plus microsomal fraction from brain
cortex incorporated P* quite rapidly and this
incorporation was stimulated by carbamylcholine.

FEDERATION PROCEEDINGS

Volume 1§

This suggests that the stimulation of P* jp.
corporation into the phospholipids of brain cortex
does not depend on the intact cell.

900. Uptake and accumulation of amino acids
by lactic acid bacteria. JosEpH T. Houpen*
AND EvuGENE Roserts. Biochemistry Dept.
Med. Research Inst., City of Hope Med. Ctr,,
Duarte, Calif.

Uptake and accumulation of amino acids by
washed cells of Lactobacillus arabinosus, Leuco-
nostoc mesenteroides, and Streptococcus faecalis
harvested from vitamin Be-supplemented or
-deficient media are under investigation employing
chromatographic, enzymatic and isotopic tech-
niques. Free (or easily extractable) amino acid
pools in these organisms are qualitatively similar
to those found in other gram-positive bacteria,
Notable constituents in L. arabinosus are y-amino-
butyric acid and p-glutamic acid. Glucose di-
minishes the depletion of the pool which occurs
during incubation in buffer alone. Glutamic acid is
accumulated in very large amounts by L. arabino-
sus and L. mesenteroides when these microorgan-
isms are incubated in buffer containing this amino
acid and glucose. The ability to concentrate this
amino acid varies markedly with the age of the
culture and is dependent on the presence of
glucose. Following exposure of L. arabinosus to
L-C!4-glutamic acid (uniformly labeled), the
accumulated, readily extractable amino acid is
found to be a mixture of the L- and p-isomers.
Vitamin Be-deficient cells accumulate relatively
greater amounts of the p-isomer, whereas the
vitamin Bes-adequate cells contain more of the
L-isomer. Only the vitamin Be-adequate cells
produce large amounts of y-aminobutyric acid
from the assimilated glutamic acid. A vitamin By
deficiency in L. arabinosus slightly diminishes the
ability to accumulate glutamic acid.

901. Free nucleotides and related compounds
of normal and infarcted human placenta.
C. R. Hoover,* A. E. WiLHELMI AND R. A.
BaRTHOLOMEW.* Depts. of Biochemistry and of
Obstetrics and Gynecology, Emory Univ., Emory
University, Ga.

To test the hypothesis that placental infarction
is of etiological significance in toxemias of preg-
nancy, the organization of free acid-soluble
nucleotides and related substances has _ been
studied in the human placenta. Immediately after
delivery of the placenta, excised samples were
compressed between slabs of dry-ice, extracted
with perchloric acid and the extracts chromato-
graphed on Dowex-1 (formate) columns. Nucleo-
tides of adenine predominated, but those of
guanine, hypoxanthine, cytosine and uracil were
easily demonstrated. Triphosphopyridine nucleo-

ee ae ae ae ee ie ee ee ae oe ae a ee ee ee a rn ee eS

olume 1§

P2 jp.
n cortex

1O acids
[OLDEN*
| Dept.,
dd. Cire

cids by
, Leuco-
faecalis
ited or
iploying
ic _tech-
no acid
similar
acteria,
-amino-
‘ose di-
. occurs
¢ acid is
vrabino-
oorgan-
s amino
ate this
of the
nce of
osus to
1), the
acid is
somers.
atively
sas the
of the
e cells
ic acid
min Bs
hes the

younds
centa.
R.. Ag
and of
Emory

arction
f preg-
soluble

been
y after
3 were
vracted
omato-
lucleo-
ose of
il were
1ucleo-

March 1956

tide could-not be detected. Material from 2 cases
of pre-eclampsia, presumably representing at
least some infarcted tissue, differed from normal
placentas. In the less severe case diminution of
adenosine triphosphate (ATP) was the only
unusual finding. In the 2nd and far more severe
case there was also near obliteration of those
substances normally eluted as cytidine mono-
phosphate, diphosphopyridine nucleotide and uric
acid, and apparently complete obliteration of
inosine monophosphate as well as some diminution
of ATP. Components not retained on formate
columns were chromatographed on Dowex-l
(Cl-) columns. All substances which were
separated with this system (free purines, pyrimi-
dines, nucleosides) were elevated in material from
the toxic cases, particularly the more severe one.
Normal term placentas were autolyzed in an
attempt to imitate extreme infarction. Study of
extracts from such preparations showed that
autolysis destroyed virtually all free nucleotides
and produced large quantities of uracil, xanthine
and hypoxanthine.

902. Role of xylulose 5-phosphate in the
transketolase reaction. B. L. HoRECKER AND
P. Z. Smyrniotis.* Natl. Insts. of Health,
PHS., Bethesda, Md.

The reaction: ribulose 5-phosphate (Ru-5-P) =
xylulose 5-phosphate (Xu-5-P) is catalyzed by
phosphoketopentoepimerase (epimerase). This
enzyme has been purified from extracts of Lacto-
bacillus pentosus and employed for the preparation
of Xu-5-P (J. Hurwitz. Federation Proc., 15:
1956). With the aid of this enzyme and Xu-5-P
the question of the specificity of transketolase has
been re-examined. Preparations of transketolase
from yeast have been found by Racker and his co-
workers (personal communication) to utilize
Xu-5-P rather than Ru-5-P. It has now been
shown that transketolase prepared from liver or
spinach, like the yeast enzyme, is unable to
utilize Ru-5-P or ribose 5-phosphate (R-5-P) for
the formation of sedoheptulose 7-phosphate
(S-7-P) and triose phosphate unless epimerase is
added. In the absence of epimerase, transketolase
is active with a mixture of Xu-5-P and R-5-P,
although neither substrate alone is utilized. The
pentose formed in the transketolase reaction with
fructose 6-phosphate and triose phosphate has
been shown to be xylulose, rather than ribulose.
It is thus apparent that the substrate for trans-
ketolase is Xu-5-P rather than Ru-5-P and that
R-5-P acts as the C-2 acceptor. Crystalline
aldolase preparations from muscle contain sig-
nificant epimerase activity which accounts for the
earlier observation (J. Biol. Chem. 205: 661, 1953)
that aldolase is required for S-7-P synthesis from
Ru-5-P and R-5-P with liver transketolase. A new

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

277

procedure for the purification of transketolase
from spinach has been developed. These prepara-
tions are more active than those previously
reported and are free of epimerase.

903. Chemical studies on new bile acids from
rat and hog bile. 8. L. Hsta,* J. T. Matscur-
NER* AND SrpNEyY A. TuHayeEr. Dept. of Bio-
chemistry, St. Louis Univ. School of Medicine,
St. Louis, Mo.

In the course of a study of normal rat bile, a
trihydroxy acid, acid I, which is slightly less polar
than cholic acid, was isolated. Subsequently, a
metabolite of labeled chenodeoxycholic acid
(MaHowaLp et al. Federation Proc. 14: 250, 1955)
was found to be identical with this acid. Quanti-
tative oxidation with periodic acid of acid I, as
well as another chromatographically similar acid
obtained from hog bile (Matscu1nEr et al. Federa-
tion Proc. 14: 252, 1955), indicated that both are
glycols. On oxidation with chromic oxide, both of
these acids gave rise to the same bilianic acid.
Hydrolysis of the bromide of methyl 3a-acetoxy-7-
ketocholanate gave a ketol which on oxidation
with chromic oxide also gave the same bilianic
acid, 3-keto-6,7-secocholanic acid-6,7-dioic acid.
Desulfuration of the ethanedithiol derivative of
this ketol gave 3a,6a-dihydroxycholanic acid.
Hydrogenation of the ketol, 3a,6a-dihydroxy-7-
ketocholanic acid using a platinum catalyst in
acetic acid, alcohol or basic solutions gave an acid
identical with that obtained from hog bile. Re-
duction with sodium borohydride gave the same
compound. The product of a Meerwein-Pondorf
reduction of 3a,6a-dihydroxy-7-ketocholanic acid
was identical with the trihydroxy acid obtained
from rat bile (acid I) and which was identified as a
metabolite of labeled chenodeoxycholic acid.

904. Squid rhodopsin. Ruta HusBarp AND
Rosert C. C. St. Georce.* Biological Labs.,
Harvard Univ., Cambridge, Mass.

Squid rhodopsin bleaches in the same steps as
vertebrate rhodopsin, but with important dif-
ferences. Like vertebrate rhodopsin, bleaching in
the squid pigment goes through the intermediate
stages, lumi- and meta-rhodopsin, to a final
mixture of retinene and opsin. Whereas in verte-
brates meta-rhodopsin is highly unstable, in the
squid it is stable at temperatures below 15°C and
at pH 6.5. Brought to px 9, it dissociates into
retinene and opsin; brought back to px 6.5, the
components reassociate to meta-rhodopsin (Sr.
GeorceE et al. Federation Proc. 11: 153, 1952). A
plot of the equilibrium concentration of meta-
rhodopsin against pH yields a titration curve with
pk 7.7. The shape of this curve indicates that the
formation of meta-rhodopsin involves the binding
of one hydrogen ion: retinene + opsin + Ht =

278

meta-rhodopsin. Squid opsin (like vertebrate
opsins) requires a specific isomer of retinene to
form rhodopsin. This differs from the isomer
present in meta-rhodopsin, which must be isomer-
ized to resynthesize rhodopsin. This is achieved
by irradiation, either of free retinene or of meta-
rhodopsin. In either case, about 4 of the retinene is
converted back to rhodopsin. Starting from pure
rhodopsin or meta-rhodopsin, irradiation yields
the identical steady-state mixture of 1 part
rhodopsin to 2 parts meta-rhodopsin. Since the
absorption spectra of squid rhodopsin and meta-
rhodopsin are nearly identical, this implies that
the photosensitivity of the 2 substances is nearly
equal. Squid opsin therefore acts as a photo-
isomerase (cf. Huspparp. J. Gen. Physiol., 1956.
In press) which in the light isomerizes retinene
combined in rhodopsin or meta-rhodopsin to a
mixture of both substances.

905. Enzymatic conversion of serine to
glycine. F. M. HurennexKeEns, Y. HatTert,*
L. Kay* ann G. Dopson.* Dept. of Biochemistry,
Univ. of Washington, Seatile.

Serine aldolase, an enzyme system which cata-
lyzes the conversion of serine to glycine, has been
partially purified from phosphate buffer extracts
of acetone-dried beef liver by means of acetone
and ammonium sulfate fractionation. The enzyme
splits L-serine into stoichiometric amounts of
glycine and a one-carbon fragment probably at the
oxidation level of formaldehyde. In the presence
of formaldehyde and formate dehydrogenases,
DPNH oxidase and catalase, the oxidation of the
one-carbon moiety can be followed manomet-
rically, thus providing a convenient assay for
serine aldolase. Sarcosine and pyruvate are inert
in this system, thereby excluding them as inter-
mediates in the conversion of serine to glycine.
The assay system requires DPN, reduced gluta-
thione (GSH), Mn**, pyridoxal phosphate (PyP)
and AT? for maximum activity. DPN is required
for the dehydrogenases. GSH is a specific cofactor
for formaldehyde dehydrogenase (STRITTMATTER
AND Bat) and cannot be replaced by other thiols.
PyP and Mn* are probably concerned with the
formation of an initial complex with serine; the
metal ion cannot be replaced by Mgt* or Cut".
There is no requirement for added tetrahydrofolic
acid in this system. ATP can be replaced by the
triphosphates of guanosine, uridine and cytidine.
Experiments with P*-labeled ATP have shown
that it phosphorylates a nondialyzable cofactor in
the system. The phosphorylated cofactor may be
released by heat denaturation and purified by
paper chromatography.

906. Release and splitting of DPN in mito-
chondria. F. Epmunp Hunter, Jr., J. LEvy,*

FEDERATION PROCEEDINGS

Volume i§

J. Davis* anp L. Caruat.* Pharmacology
Dept., Washington Univ. School of Medicine,
St. Louis, Mo.

The loss of DPN from rat liver mitochondria
during preincubation with 0.02 m phosphate ip
0.25 m sucrose is not the result of simple diffusion
from swollen mitochondria. Suspension of mito-
chondria in markedly hypotonic media results in
equal swelling with no loss of DPN. Three washes
with 0.0125 m sucrose or mannitol (5% of isotonic)
result in greater swelling than ever produced by
phosphate, yet most of the endogenous DPN
(6-hydroxybutyrate oxidation) remains intact. In
contrast, 3 washes with water, 0.25 m urea, or
0.0125 m urea or glycine result in loss of nearly all
of the DPN. The protective effect of even low
concentrations of sucrose and mannitol may be
related to osmotic effects. During preincubation of
mitochondria with phosphate the DPN is split.
This split appears to be almost entirely at the
nicotinamide riboside bond, as there is close
correlation between determinations by fluo-
rescence (dependent on nicotinamide riboside
link) and by enzymatic methods dependent on the
whole molecule. Added DPN is also split at the
nicotinamide riboside link. The rate of splitting is
similar with control mitochondria and those
swollen by pretreatment with phosphate. Nico-
tinamide inhibits almost completely the splitting
of both endogenous and added DPN, but is with-
out effect on the swelling of mitochondria. Sub-
stances which do prevent swelling and loss of
endogenous DPN (EDTA, Mnt*, Mgt’, ADP,
ATP) do not inhibit splitting of added DPN.
(Supported by a grant from the Natl. Insts. of
Health, PHS.)

907. Enzymatic interconversion of ribulose
5-phosphate and xylulose 5-phosphate.
J. Hurwitz (introduced by H. G. Sternman).
Natl. Insts. of Health, PHS, Bethesda, Md.
Ashwell and Hickman (J. Am. Chem. Soc. 76:

5889, 1954) identified a phosphate ester of p-

xylulose in spleen preparations incubated with

ribose 5-phosphate (R-5-P). The formation of this
product can be attributed to an enzyme which
catalyzes the reaction: p-ribulose 5-phosphate

(Ru-5-P) = p-xylulose 5-phosphate (Xu-5-P). This

enzyme, phosphoketopentoepimerase (epimerase),

is present in extracts of Lactobacillus pentosus

(StumpF aND Horecker. J. Biol. Chem., Feb.

1956). It has been purified more than 500-fold

from this source; these preparations are free of

transketolase and phosphoriboisomerase (isomer-
ase). At 25°, in the presence of purified bacterial

epimerase, an equilibrium mixture containing 60%

Xu-5-P and 40% Ru-5-P is formed. Xu-5-P, free

of R-5-P and Ru-5-P, has been prepared from

R-5-P by the action of epimerase and spinach

olume 1§

macology
ledicine,

shondria
hate in
liffusion
of mito-
sults in
> washes
sotonic)
uced by
is DPN
tact. In
irea, or
arly all
ven low
may be
ation of
is split.
at the
is close
y fluo-
riboside
t on the
, at the
itting is
| those
. Nico-
plitting
is with-
a. Sub-
loss of
. ADE

DPN.
ists. of

bulose
phate.
NMAN),
:
oc. 76:
of p-
d with
of this
which
sphate
). This
erase),
ontosus
, Feb.
00-fold
free of
somer-
cterial
1g 60%
P, free
| from
pinach

March 1956

isomerase. The sugar component has been identi-
fied as D-xylulose by its optical rotation (a? =
—38.5°) and by chromatography of the polyols
following borohydride reduction. The phosphate
group has been shown to be located in the C-5
position by periodate oxidation.

908. Erythrocytin, a clotting factor from
erythrocytes: its action and purification.
Ciara V. Hussey anp Maraaret M. Kaser
(introduced by JoserpH C. Bock). Dept. of
Biochemistry, Marquette Univ., and VA Hosp.,
Milwaukee, Wis.

An extract of hemolyzed erythrocytes can
replace the platelets in the clotting reaction of
blood. When erythrocyte extract is added to
normal platelet-poor plasma, the prothrombin
consumption is greater than when a platelet
extract is used. It can also adequately replace
platelets in the thromboplastin generation test.
The active factor is in the stroma which permits
its separation of hemoglobin. The hemolyzed
erythrocytes are diluted 10-fold with saline
solution and centrifuged at 5000 rpm for 15 min.,
whereupon the stroma mass is separated from the
soluble hemoglobin. The product from the stroma
is nearly colorless and has high activity as meas-
ured by the prothrombin consumption test but no
longer has activity as determined by the thrombo-
plastin generation test. Further purification was
effected by dehydrating the stroma with acetone
and then extracting it with chloroform. After
removing the solvent, a small quantity of an
amorphous material was obtained which, after it
was homogenized in physiological saline, had a
high degree of activity. The name ‘erythrocytin’
is proposed for this clotting factor. (Supported
by a grant from the Public Health Service.)

909. Growth stimulation of Streptococcus
faecalis by adenine derivatives. Dorris J.
Hutcuison (introduced by AARoN BENDICH).
Sloan-Kettering Inst. for Cancer Research, New
York City.

The ability of certain antimetabolite-resistant
strains of Streptococcus faecalis to grow on adenine,
adenosine and adenylic acids has been determined.
The microorganisms used in this study were S.
faecalis 8043 and several strains resistant to
amethopterin, 6-mercaptopurine, and 2,6-di-
aminopurine. In a thymine-containing medium,
adenine and adenosine were good growth-pro-
moting substances for all these microorganisms
with the exception of one 6-mercaptopurine-
resistant strain. (Incorporation experiments with
labeled adenine and adenosine with this strain
show the utilization of the adenine moiety for
nucleic acid adenine but not nucleic acid guanine.)
Adenosine-5’-phosphate and adenosine-2’-phos-

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

279

phate did not support the growth of any of these
microorganisms, while adenosine-3’-phosphate
was capable of supporting the growth of all with
the notable exception of the 6-mercaptopurine-
resistant strain. Two of the antifolic-resistant
strains cannot grow in a folic acid-containing
medium but can grow when any of the adenine-
containing compounds are added to the medium
The addition of these adenine compounds to the
folic acid medium does not stimulate the growth
of any of the other microorganisms. The growth
data suggest that the adenine moiety from the 3
adenylic acids is available as a growth-promoting
agent to these antifolic-resistant strains and may
serve in a role which is not concerned directlv
with nucleic acid synthesis.

910. Phosphate content of Ehrlich’s ascites
tumor cells and its importance in metabo-
lism. KENNETH Ipsen,* BarsBara McCarty*
AND Rautpu W. McKes. Dept. of Physiological
Chemistry, Univ. of California, Med. Cir., Los
Angeles.

The authors (Cancer Research 13: 537, 1953) have
observed that the in vitro addition of small
amounts of glucose (9-18 mg %) stimulated the
oxygen consumption of washed Ehrlich’s ascites
tumor cells, while the addition of larger quantities
of glucose (50-150 mg %) inhibited oxygen utiliza-
tion by the tumor cells during the first hour of
respiration. In addition, it has been observed
that the glucose content of the tumor present in
mice is low (0-10 mg %). Furthermore, the tumor
grows more slowly in obese-hyperglycemic and
alloxan-diabetic mice and the host animal survives
longer (Cancer Research 15: 341, 1955). Preliminary
in vitro experiments indicated that the inhibition
produced by 50 mg % glucose was reduced 25-50%
by the addition of 15-55 ma/I. of inorganic phos-
phate. Further studies have indicated that
increasing the phosphate stimulated, over a 3-hour
period, the oxygen consumption of the cells.
Analyses of the tumor cells for the various phos-
phates show that the inorganic phosphate is
particularly high (7-day tumor, 11-15 mm/lI.).
The mechanism for the inhibition of oxidative
metabolism with excessive amounts of glucose is
not clear. However, Chance et al. (J. Biol. Chem.
217: 383, 1955) suggests that there may be a
competition by the oxidative and glycolytic
systems for ADP. Work is in progress to develop
an appropriate cell or cell particulate system for
testing the influence of various organic phosphates
on oxygen consumption in the presence of glucose.

911. Inhibition by sulfathiazole of the bio-
synthesis of 5-amino-4-imidazolecarboxa-
mide (AICA) by Escherichia coli, strain
B96. Kanro Isu1* anp M. G. Srvaa. Dept. of

280

Microbiology, School of Medicine,

Pennsylvania, Philadelphia.

When sulfathiazole inhibits the growth of wild
strains of EL. coli there is a concomitant stimula-
tion of the accumulation of AICA. However, we
have observed that sulfathiazole inhibits both the
growth and the accumulation of AICA by a
purine-requiring FZ. coli mutant (B-96) in a casein
hydrolysate-glucose-adenine medium. AICA ac-
cumulation by B-96 is also inhibited by sulfathia-
zole in a nongrowing system lacking adenine and
in a sulfathiazole-resistant strain derived from
B-96. This inhibition is not antagonized by p-
aminobenzoic acid. It can be concluded from the
following evidence that inhibition of AICA
accumulation by sulfathiazole is due to its effect
on the utilization of glutamic acid for AICA bio-
synthesis. Amino acids must be present in order
for this inhibition to occur, as no inhibition of
AICA accumulation occurs in a salts-glucose
resting system. The amount of AICA formed in
casein hydrolysate-glucose medium is greater than
that formed in salts-glucose medium, so that the
actual effect of sulfathiazole is to counteract the
contribution by amino acids to the accumulation
of AICA. Of all the combinations of amino acids
tested, serine and glutamic acid produce the
greatest amount of AICA. When serine is the sole
nitrogen source, sulfathiazole does not inhibit the
production of AICA. However, sulfathiazole does
inhibit the AICA produced from glutamic acid.
The utilization of serine is not depressed by
sulfathiazole, whereas glutamic acid utilization is
inhibited by sulfathiazole.

Univ. of

912. Reaction of blue tetrazolium with C21
and C19 steroids. ANTHONY J. Izzo, RoBERT
B. Burton anp E. Henry KevuTMANN (intro-
duced by Frances Haven). Dept. of Medicine,
Univ. of Rochester School of Medicine and Den-
tistry, Rochester, N. Y.

The ,hemical reduction of blue tetrazolium
(BT) by steroids possessing an alpha ketol side
chain has been used for both their qualitative
detection and quantitative estimation. Although
some of the characteristics of the reaction have
been established by several investigators, there
has been some disagreement on the precise con-
ditions that will allow optimal as well as selective
reduction by the ketolic steroids. The influence of
different concentrations of alkali on the speed and
extent of reduction of blue tetrazolium by various
C21 and C19 steroids was studied over a period of
2 hr. at 25°C. The range of concentration of
tetramethyl ammonium hydroxide tested in the
reaction mixture was from 0.0022 N-0.22 N. When
a concentration of 0.022 N was used all ketols
reacted at the same rate and to the same extent
between 30 and 60 min. The differences observed

FEDERATION PROCEEDINGS

Volume 1§

were minimal and probably not significant. Non-
ketolic steroids with a conjugated 3-keto group
caused much less reduction of BT and at a much
slower rate. The rate was also influenced by the
concentration of alkali but was always much
slower than with ketols. When the concentration
of base in the reaction mixture was 0.022 N the
rate was a straightline function up to 60 min,
Addition of an 11 and/or 17 keto group enhanced
the reaction of the steroids with a conjugated
3-keto group. Addition of a 6-beta-hydroxy or
6-keto group decreased the reactivity of these
compounds. An oxygen function at carbon 3, II,
12, 17, or 20 was unreactive unless the delta 4,
3-keto group was also present. Mixtures of ketolic
steroids with slowly-reacting non-ketolic steroids
gave reduction equivalent to the sums of the
separate compounds. When neutral urine extracts
were reacted for 30 min. at 25°C and a concentra-
tion of base of 0.022 N in the reaction mixture,
the relationship of amount of urine extract to the
optical density reading followed Beer’s law. Con-
tinuation of the reaction beyond 30 minutes caused
a gradual linear increase of reduction, suggesting
the presence of some non-ketolic substances. Since
the ketols in the extracts reacted maximally by
30 min., correction could be made for the presence
of slow-reacting non-ketolic material by measuring
the rate of reaction beyond 30 min. Addition of
slowly-reacting pure non-ketolic steroids to urine
extracts could be accounted for quantitatively by
making such a correction. Recovery of added pure
ketolic steroids to the urine extracts was quanti-
tative.

913. Phosphorylation coupled to oxidation
and reduction of silico-molybdate in rat
liver mitochondria. Earut E. Jacosps* Anp
D. R. Sanapt. Inst. for Enzyme Research, Univ.
of Wisconsin, Madison, and Dept. of Physiological
Chemistry, Univ. of California Med. School,
Berkeley.

The oxidation of ascorbate in rat liver mito-
chondria is relatively slow (Lardy, Lehninger).
The oxidation rate is increased by the addition of
external cytochrome C but the P/O is decreased
under these conditions. We have found that
catalytic quantities of silico-molybdate (Ey’ =
+0.45) will cause mitochondria to oxidize
ascorbate at rates comparable to those observed
with succinate as substrate. The P/O ratios
consistently approach 1.0. Removal of bound
cytochrome C from mitochondria completely stops
the oxidation even in the presence of silico-
molybdate. This suggests that the observed
phosphorylation is associated with electron
transport via the cytochrome oxidase system.
Silico-molybdate is reduced rapidly by succinate
in the presence of saline-washed cytochrome

—- = oo «

a ee a n,n ee ee

—-_

olume 16

it. Non-
O group
a much
1 by the
S much
ntration
2 N the
60 min,
nhanced
jugated
roxy or
of these
n 3, Il,
delta 4,
ketolic
steroids
of the
extracts
icentra-
nixture,
t to the
w. Con-
3 caused
gesting
s. Since
ally by
resence
asuring
ition of
O urine
vely by
ed pure
quanti-

dation
in rat
3* AND
, Univ.
ological
School,

* mito-
inger).
tion of
reased
1 that
‘Ey’ =
oxidize
served
ratios
bound
y stops
silico-
served
ectron
ystem.
-cinate
hrome

March 1956

C-free mitochondrial residue. The possibility of
phosphorylation coupled to this reaction is under
investigation.

914. Amino acid absorption from the small
intestine by continuous flow perfusion in
situ. Francis A. Jacoss, M. Luper anp E. V.
GILBERTSON (introduced by D. F. Eve etn).
Dept. of Biochemistry, Univ. of North Dakota
School of Medicine, Grand Forks.

The absorption of glycine by the upper segment
of the small intestine of the rat has been studied in
an in situ perfusion technique. A new perfusion
method has been designed in which continuous
intestinal absorption studies can be carried out
on an intact animal. Adult male rats, employed
in all instances, were fasted for 18 hr. and then
anesthetized with sodium pentobarbital. Known
amounts of glycine in the solvent under investi-
gation were perfused through the intestinal
segment at the rate of 1-2 ml/min. Samples (0.2
ml) were withdrawn from the system at intervals
of 5, 15, 30, 45 and 60 min. after the zero time of
the perfusion period. It was found that the uptake
rate of glycine dissolved in Krebs-Ringer-Phos-
phate buffer followed similar patterns over the
concentration range from 0.02-0.08 molar, as
measured by its disappearance from the perfusate.
This pattern was found to be the same for the
amino acid carried in physiological sodium chloride
solution. However, there was variability in the
uptake in extremely hypotonic solutions, i.e. the
amino acid at 0.02-0.08 m concentrations in water
solution. This latter finding is apparently related
to the marked loss of water across the intestinal
mucosa with these hypotonic solutions.

915. Factors influencing intracellular pyri-
dine nucleotides. K. Bruce JACOBSON (intro-
duced by E. V. McCotium). McCollum-Pratt
Inst., Johns Hopkins Univ., Baltimore, Md.

A pyrophosphatase from pigeon liver was
purified that splits reduced DPN but not oxidized
DPN. The enzyme also splits FAD, adenosine
diphosphate ribose, reduced analogs of DPN but
not the oxidized analogs. After differential
centrifugation of pigeon liver the DPNH pyro-
phosphatase appeared in the supernatant from
105,000 X g; another pyrophosphatase was de-
tected in the nuclear and microsomal fractions
which split both DPN and DPNH. The mito-
chondrial fraction contained almost no splitting
activity on either oxidized or reduced DPN. In
these characteristics fractionated rabbit liver
resembled pigeon liver whereas the rat, mouse and
hamster livers differed in that the supernatant had
little or no capacity to destroy either DPN or
DPNH. The mitochondrial fractions of all 5
animals had very little pyrophosphatase activity.

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

281

The distribution of DPNase (nicotinamide ribose
cleavage) closely paralleled that of DPN pyro-
phosphatase. The distribution of DPN was
assayed in liver fractions. Over 50% of the rat
liver homogenates endogenous DPN appeared in
the mitochondria. The DPN of whole homogenates
and mitochondria increased markedly on storage
at 0-3°. We have demonstrated that a nicotinamide
ribose split of mitochondrial DPN occurs even
though less than 2% of the total DPNase activity
appeared in the mitochondria. Versene inhibited
this splitting (HUNTER AND Forp, J. Biol. Chem.
216: 357, 1955) although it failed to inhibit similar
destruction of exogenous DPN. The disappearance
of mitochondrial DPN was not stimulated by
added Neurospora DPNase suggesting that DPN
must be released from the mitochondria prior to
destruction.

916. Conversion of $-carbon of serine to
N.-formyltetrahydrofolic acid. L. JAENICKE
(introduced by 8S. Levey). Dept. of Biochemistry,
Western Reserve Univ., Cleveland, Ohio.

The 8-carbon of serine is transferred to tetra-
hydrofolate (FAH,) and is oxidized to the formyl
group of N°-formyltetrahydrofolate (N?-
CHOFAH,) (JaEnicke. Biochim. et Biophys Acta.
17: 1955, 588). The enzyme system from pigeon
liver has been fractionated into two overall steps
which tentatively may be formulated as: (1)
serine + FAH, = FAH,-CH.OH + glycine;
(2) FAH,-CH,OH + TPN = N”-CHOFAH, +
TPNH. Reaction / requires Mn** and pyridoxal-
phosphate. In the second reaction, DPN showed
only about one tenth the activity of TPN. The
oxidation step was inactivated at 58°, whereas
the condensation step / was stable. FAH,-CH,OH
produced by enzyme / was isolated by paper
chromatography. It showed an absorption maxi-
mum at 270 my and gave the CHO reaction. By
enzyme (2) 80% of FAH,-CH:OH was converted
to N”-CHOFAH,. CH:0 reacted with FAH,
non-enzymatically to form a hydroxymethyl
tetrahydrofolate compound which also was
oxidized by enzyme 2 to N°-CHOFAH,. Fraction
1, treated with charcoal to remove pyridoxal-
phosphate and fractionated further, condensed
serine and FAH, to a folic acid derivative, X,
which could be separated by paper chroma-
tography. The compound was yellow, showed a
blue fluorescence on paper, possessed one ab-
sorption maximum at 272 my and gave a ninhydrin
color. Acid hydrolysis of the compound formed
glycine and a product which reacted as formalde-
hyde. X with the unfractionated system in the
absence of TPN was converted to equimolar
quantities of glycine and a compound giving the
CH:0 reaction. In the presence of TPN, X was
converted to N°-CHOFAH, and glycine.

282

917. Enzymatic decarboxylation of oxalic
acid. Wiitu1aAM B. Jakosy (introduced by I.
Zeuitcn). Natl. Insts. of Health, Bethesda, Md.
A partially purified enzyme system has been

obtained from a bacterium isolated from soil

which will catalyze the anaerobic decarboxylation
of oxalic acid. ATP, CoA, magnesium ions, acetate
and thiamine pyrophosphate (ThPP) are required
for decarboxylation. When acetyl CoA is substi-
tuted only ThPP is required. The data are in
accord with the following mechanism: /) transfer
of CoA from acetyl CoA to oxalate to form oxalyl
CoA; 2) decarboxylation of oxalyl CoA in the
presence of ThPP. The decarboxylation of oxalic
acid in this system results in the formation of
equimolar quantities of carbon dioxide and
formate. The addition of DPN to the system
results in the formation of two moles of carbon
dioxide per mole of oxalate utilized due to the
presence of a DPN-specific formic dehydrogenase.

918. Intracellular distribution and metabolic
heterogeneity of ribonucleic acid (RNA) in
regenerating liver. CHRISTINE D. JARDETZKY*
AND Cyrus P. Barnum. Dept. of Physiological
Chemistry, Univ. of Minnesota Med. School,
Minneapolis.

The amount of RNA per gram fresh liver was
measured quantitatively on trichloroacetic acid
hydrolysates of tissue using Bial’s method for
ribose, and the metabolic activity of the phos-
phorus of purified RNA samples was determined
by the incorporation of P*? at different times
after the injection. The mice were killed at times
varying from 0.5-6 hr. after the intraperitoneal
injection of P%? and 58-64 hours after partial
hepatectomy. The livers were homogenized in
alkalinized physiological saline and the resulting
homogenates were fractionated by differential
centrifugation into nuclear (N), mitochondrial
(L), microsomal (M), ultramicrosomal (U), and
super#atant (S) fractions. The nuclei were further
purified with 2% citric acid. Approximately 43%
of the total RNA per gram of liver could be
accounted for by M-RNA and 6, 7, 7, and 9% by
N, L, U, and S-RNA respectively. The N-RNA
had the highest and the M-RNA the lowest
specific activity at any time during the 6-hr.
period of the kinetic study. The specific activity
of L-RNA observed at 0.5 and 1 hr. after P*?
administration was very similar to that of
M-RNA. The U fraction, composed of particles
which may be comparable to the small particulate
component of the cytoplasm recently described by
Palade (J. Biophys. and Biochem. Cytol. 1: 59,
1955), was characterized by an RNA witha specific
activity-time curve intermediate between those
of M and 8-RNA. The results confirm the intra-
cellular heterogeneity of RNA phosphorus in the

FEDERATION PROCEEDINGS

Volume ij

regenerating mouse liver and suggest the existenge
of two metabolically distinct RNAs associated
with the cytoplasmic particulate fractions, the J
and M-RNA.

919. Color reaction of N-acetylamino sugar
derivatives with p-dimethylaminobenzalde.
hyde. Roger W. JEANLOZ AND Moniqup
Trémece.* Lovett Mem. Lab., Massachusetts
Gen. Hosp., and Dept. of Biological Chemistry,
Harvard Med. School, Boston.
N-acetylglucosamines linked in position 4 haye

been shown not to react with p-dimethylamino.

benzaldehyde (Morgan-Elson reaction) (Kuny,

GAUHE AND Barr. Chem. Ber. 87: 1138, 1954). Ih

order to study the influence of substituents on

the amount of color produced, methylated deriva-
tives of N-acetylglucosamine and N-acetyl
galactosamine have been analyzed according to

Aminoff, Morgan and Watkins (Biochem. J. 51:

379, 1952). Compared to N-acetyl-p-glucosamine,

the values obtained were: 3-methyl- 160%;

4-methyl- 3%; 6-methyl- 100%; 3,4-dimethyl- 3%;

3,6-dimethyl- 140%; 4,6-dimethyl- 8%; 3,4,6.

trimethyl-N-acetyl-p-glucosamine 1%; N-acetyl-

p-galactosamine 25%; 4-methyl- 4%; 3,4

dimethyl- 2%; 4,6-dimethyl- 3%; 3,4,6-tri-

methyl-N-acetyl-p-galactosamine 2%; N-acetyl
p-allosamine 50%. In the limit of error of the
method, it can be shown that substitution in
position 4 prevents the color formation; substitu-
tion in position 6 does not affect it, and substitu-
tion in position 3 increases it approximately by

50%. The influence on color formation of the

configuration of the hydroxyl groups at positions

3 and 4 is also shown by the results obtained with

N-acetylgalactosamine and N-acetylallosamine.

Application of such color reactions simplifies

differentiation of methylated aminosugars isolated

in minute amounts by paper chromatography.

However, it is inadequate for following the

degradation of N-acetylhexosamine-containing

substances when the positions of the substituents
are not known.

920. Turnover rates of serum protein of
normal dogs. HENRY JEFFAY AND SAMUEL
Mozersky (introduced by M. E. Raretson).
Univ. of Illinois College of Medicine, Chicago.
The turnover rates of the electrophoretic serum

protein fractions of dogs were determined by two

methods. In one series, radioactive L-methionine-

S*5 was administered and the specific activities of

the 6 individual electrophoretic components were

determined at various intervals after separation
by paper electrophoresis. The half-lives of the
albumins were constant over a 40-day period

(t; = 19-30 days). The gamma globulin fraction

had half-lives of 13-18 days, which were also

ee eRe gee OE ae eee ae Loe Se ee Re re ge ee ee

ee ee ee ee ee ee ee ee

ee Ey tay ee a ee a ee ee aT eee ork ee

Volume ij

existence
ssOciated
ns, the J

10 sugar
enzalde.
Monique
sachusetts
‘hemistry,

m 4 have
ylamino-

(Kunn,
1954). In
uents on
d deriva.
N-acetyl.
rding to
n. J. oF
osamine,
- 160%;
hyl- 3%;
> 3,4,6-
V -acetyl-
0; 3,4
3,4, 6-tri-
v-acetyl-
of the
ution in
substitu-
substitu-
ately by
1 of the
rositions
ned with
samine.
implifies
isolated
graphy.
ing the
ntaining
stituents

tein of
SAMUEL
‘ELSON),
vicago.
ic serum
| by two
hionine-
vities of
its were
yaration
of the
period
fraction
re also

March 1956

reasonably; constant as a function of time. The
a'pha and beta globulins, however, showed a much
shorter half-life (5-6 days) for the first week, but
following this period, the half-lives were pro-
longed. These results suggested that, when the
specific activity of the alpha and beta fractions
fell significantly below that of the albumin and
of the body amino acid pool, reincorporation of
the isotope into these fractions proceeded at a
rapid rate. In the other series, S*-labeled serum
albumin isolated by starch slab electrophoresis
was administered to normal dogs. The half-life of
albumin was approximately 11 days. The specific
activity of the total globulin fraction was initially
very low but rose to about one-tenth that of the
albumin fraction, followed by a decay rate corre-
sponding to a half-life of about 20 days. These
results indicate that albumin may be converted
either directly or via the free amino acid pool to
other serum proteins, and that re-utilization of
isotope gives a spuriously long half-life to those
protein fractions which turn over at a rapid rate.

921. Transamination of certain aliphatic
amino acids by an enzyme from chicken
liver. W. Terry JENKINS* AND Irwin W.
Sizer. Div. of Biochemistry, Dept. of Biology,
Massachusetts Inst. of Technology, Cambridge.
The conversion of alpha hydroxy acids to amino

acids in a preparation from chicken liver involves
oxidation to the keto acid followed by transamina-
tion. Purification of the enzyme responsible for
the transamination from leucine to form methio-
nine has shown it to have a wide specificity.
Substrates for the enzyme include the natural
amino acids leucine, isoleucine, valine, methio-
nine, aminobutyric acid and glutamic acid.
Transamination occurs between any amino acid
and any keto acid in the same series. Since the
rate of transamination with glutamic acid is
usually less than that from another amino acid in
the series, it is believed that transamination
occurs without the obligatory formation of
glutamic acid. This, together with the fact that
the rate of transamination for 2 amino acids
together to the same keto acid is not the sum of
their individual rates, leads us to postulate a
single enzyme, analogous to the £. coli trans-
aminase B of Rudman and Meister (J. Biol.
Chem. 200: 591, 1953). Inhibition by heavy metals,
iodoacetate, p-chloromercuribenzoate and iodoso-
benzoate, indicates that thiol groups are necessary
for enzymatic activity. Purification in the absence
of versene yields preparations which can be
activated by glutathione, cysteine, or versene. The
enzyme is not activated by preincubation with
pyridoxal phosphate. The px for optimum activity
is greater than pH 8 but the enzyme is markedly
unstable in the alkaline range.

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

283

922. Thrombin formation. H. JENSEN AND
Arnotp B. Tre1n.* Army Med. Research Lab.,
Fort Knoz, Ky.

In the coagulation of blood the conversion of
prothrombin to thrombin is preceded by the
formation of a prothrombin converting factor. The
present investigation concerns itself with the
intermediate reaction involving the formation of
a prothrombin activator prior to the conversion of
prothrombin to thrombin. Preincubation of a
BaSO,-eluate from rabbit serum with platelets and
CaCl. in the presence of plasma accelerator
globulin (PAcG) or of antihemophilic factor
(AHF) or of both PAcG and AHF before addition
of the prothrombin source to the preincubation
mixture resulted in a marked enhancement in the
prothrombin conversion as compared with a one-
stage thrombin formation procedure. The greater
velocity of thrombin formation in the 2-stage
procedure indicates that precursory interactions
of certain factors had been initiated or completed
during the preincubation. Preincubation of either
platelets, AHF and CaCl, or of platelets, PAcG
and CaCl: or of platelets, PAcG, AHF and CaCl,
before addition of the prothrombin source plus
serum eluate to the preincubation mixture resulted
in approximately the same rates of thrombin
elaboration as found in the one-stage thrombin
formation procedure. These findings indicate that
no interaction between the various factors had
taken place during the preincubation period under
these conditions. It appears from these observa-
tions that PAcG as well as AHF will interact with
platelets and calcium ions only in the presence of
factor(s) in serum eluate yielding prothrombin
converting activity.

923. P*2. incorporation into acid soluble
nucleotides contained in a virus infected
tissue. R. Bernat JOHNSON (introduced by
W. Wixtspur AcKERMANN). Dept. of Epidemi-
ology and Virus Lab., Univ. of Michigan, School
of Public Health, Ann Arbor.

While it has not been possible to detect dif-
ferences in the amounts of polymerized nucleic
acid in virus-infected and uninfected cells (JoHN-
SON AND ACKERMANN, Federation Proc. 12: 226,
1953), recent investigations of the nucleic acid
precursors in normal chorio-allantoic membranes
and in those infected with influenza virus have
revealed certain differences which are indicative
of an altered nucleic acid metabolism in this virus-
infected tissue. The nucleotides contained in
these tissues have been isolated by perchloric acid
extraction, separated by the technique of gradient
elution fractionation and measured by their
optical density at 260 my. Infected tissues which
are producing virus maximally appear to contain
the same nucleotides as the normal tissue. Radio-

284

active phosphorus has been used to label the
nucleotides in similarly handled membranes.
Measurement of the amount of P** in the fractions
has revealed the presence of phosphorus-con-
taining compounds previously not detected in
this tissue. The specific activities (radioactivity/
nucleotide content) of the nucleotides indicate
that the incorporation of P*? into certain nucleo-
tides proceeds at a faster rate in the infected
tissue than in the uninfected tissue.

924. Antimetabolite activity of 6-amino-
nicotinamide. WILLARD J. JOHNSON AND J. D.
McCot. (introduced by O. F. DeEnstept).
Research Labs., Frank W. Horner Limited,
Montreal, Quebec, Canada.

The biological activity of 6-aminonicotinamide
as a nicotinamide antagonist has recently been
reported (Science 122: 834, 1955). It has been
confirmed that tryptophan, as well as nico-
tinamide and nicotinic acid, protects mice against
the lethal effects of 6-aminonicotinamide. The
simultaneous intraperitoneal administration of
tryptophan (250 mg/kg) and 6-aminonicotinamide
doubled the dso of the latter. The 6-amino-
nicotinic acid produces effects in mice similar to
those of the amide, but only when given at ap-
proximately 15 times the dose level. This would
indicate a low rate of conversion of acid to amide
in vivo. The 6-aminonicotinamide analogue of
DPN has been isolated in good yield from a re-
action mixture containing pig brain DPN ase,
DPN, and 6-aminonicotinamide. The analogue has
been detected, by chromatographic and spectro-
photometric techniques, in liver and kidney of
mice treated with 6-aminonicotinamide, and in
neoplastic tissue from rats bearing Walker carcino-
sarcoma 256. The analogue is inactive in the yeast
alcohol dehydrogenase system, but does form an
addition compound with cyanide. The 6-amino-
nicotinamide exhibits a cumulative effect in rats.
Subcut®neous injection of 2 mg/kg of body weight
per day for 10 days resulted in 50% mortality on
the 11th day, compared with the acute LD50 of
35 mg/kg in the mouse. Walker tumor-bearing
rats appeared to withstand the lethal effects of
the antimetabolite better than did non-tumor
bearing controls.

925. New procedure for preparing salts of
heparin. Rospert L. Jones (introduced by
D. W. MacCorquopate). Abbott Labs., North
Chicago, Ill.

The n-heptylamine salt of heparin, as an
example, is prepared from the sodium salt. By
dialyzing this, or by going through the ammonium
salt, a virtually ash-free derivative of heparin can
be made. Other amines can be used. To an 80%
alcohol solution of the amine salt an 80% alcohol
solution of the desired metallic salt is added in

FEDERATION PROCEEDINGS

Volume 1§

adequate amounts. The resultant salt of heparin,
insoluble in this concentration of alcohol,
separates readily. The procedure is relatively
simple, and limited in application only by the
solubility of the inorganic salt in 80% alcohol,
The important features of the method are: 1) the
preparation of single rather than mixed salts;
2) the preparation of a variety of salts by the same
procedure.

926. Properties of threonine aldolases,
Marvin A. KaraseK* AND Davip M. GREEN-
BERG. Dept. of Physiological Chemistry, Univ. of
California School of Medicine, Berkeley.
Evidence was obtained for the existence of two

enzymes decomposing the diastereoisomers of
threonine to glycine and acetaldehyde, the ac-
tivity against L-allothreonine being the highest
and the activity against L-threonine being very
weak. Employing a partially purified sheep liver
enzyme preparation and p,t-allothreonine or
D,L-threonine as substrate it was found that
allothreonine splitting was increased 2-fold by
10-5 m pyridoxal and 20-fold by the same concen-
tration of pyridoxal phosphate, while the de-
composition of threonine was not altered. No
increase in activity with either substrate was
produced by metal ions and no loss in activity was
caused by incubation with 2,2’-bipyridine,
ethylenediaminetetraacetic acid, 8-hydroxyquino-
line, 2,3-dimercaptopropanol, glutathione nor
ascorbic acid. Cysteine, homocysteine or Cu**
were inhibitory. A compound tentatively identi-
fied as threonine was synthesized from acetalde-
hyde and glycine upon incubation with the enzyme
preparation.

927. Electrochromatography of large parti-
cles: preliminary studies with poliomyelitis
virus and tobacco mosaic virus. ARTHUR
KaRLER (introduced by Paut L. Krrx). Karler
Labs., Berkeley, Calif.

A series of investigations involving the develop-
ment and evaluation of a variety of electro-
chromatographic (continuous-flow curtain electro-
phoresis) apparatuses has been carried out. This
paper will describe some applications to prepara-
tive isolation of those types of very large
molecules or particles found in such preparations
as whole cell homogenates. Viruses were selected
as convenient model particles for testing the
method. The electrochromatographic processing
of 2 types of viruses was performed: 1) spheres as
represented by poliomyelitis virus and 2) rigid
rods by tobacco mosaic virus. Both the vertical-
and horizontal-curtain types of electrochromato-
graphic apparatus were employed. Poliomyelitis
virus (type III) was successfully isolated directly
from its culture medium without pretreatment.
Excellent separation was achieved from kidney

lume 15

1eparin,
alcohol,
latively
by the
alcohol,
: 1) the
1 salts;
he same

olases,
GREEN-
Jniv. of

of two
ers of
the ac-
highest
ig very
p liver
ine or
d that
old by
oncen-
he de-
d. No
te was
ity was
ridine,
‘quino-
e nor
> Cut*
identi-
atalde-
nzyme

parti-
yelitis
RTHUR
Karler

velop-
lectro-
lectro-
. This
epara-
large
ations
lected
g the
essing
TES a8

rigid
rtical-
mato-
yelitis
rectly
ment,
‘idney

March 1956

proteins, phenol red in the medium, and other
contaminants of the virus. After saturation of the
curtain, the live virus was obtained in good yield
and satisfactory concentration. Tobacco mosaic
virus as a representative rigid rod type presented
a more difficult problem than the spherical types
of virus. A small-scale horizontal-curtain type of
electrochromatographic apparatus proved helpful
because of the much shorter pathway, along with
resolving power comparable to that of the larger
machines. This approach to large ‘molecule’
separations has several interesting biochemical
implications: 1) the possibility of effective evalua-
tion of the electrophoretic homogeneity of pure
viruses; 2) the ease of removing trace amounts of
intact virus; and 8) the possibilities for controlled
fragmentation of the intact virus on the curtain
with subsequent fractionation of the breakdown
products. These factors are of possible significance
to the problem of virus reconstitution.

928. Synthesis and metabolism of enantio-
morphic forms of glycerol-l-C. M. L.
Karnovsky, G. Hauser* anp D. Etwyn.*
Dept. of Biological Chemistry and the Biophysical
Lab., Harvard Med. School, Boston, Mass.

D- and t-serine-8-C'™ were individually con-
verted to glycerol by deamination to glyceric acid
and reduction of diacetylethylglycerate with
lithium aluminum hydride. Glycerol obtained
from D-serine-3-C'* could be termed p-glycerol-
3-C™; that from the L-amino acid, L-glycerol-3-C".
However, it is preferable to designate the labeled
carbon as carbon atom one, and to refer to these 2
compounds as L-glycerol-1-C"* and p-glycerol-1-C"4
respectively. These substrates were incubated
with rat liver slices in Krebs-Ringer phosphate
medium. Barium glycerophosphate was isolated
from the phosphatides of the slices after alkaline
hydrolysis. In experiments with p-glycerol-1-C%,
a-glycerophosphate had 6% of its activity in the
free primary carbinol carbon; in experiments with
L-glycerol-1-C™, 96% was in this carbon. Glucose
isolated from the medium after incubation with
p-glycerol-1-C had 40% of its activity in C-1,
48% in C-6, 6% in C-3 and 8% in C-4. Conversely,
when L-glycerol-1-C' was used 8% of the glucose
activity was in C-1, 5% in C-6, 37% in C-3 and
50% in C-4. The results are compared with those
obtained with synthetic a-C'-glycerol (DL-
glycerol-1-C!4) (GiprEz AND Karnovsky. J. Am.
Chem. Soc. 74: 2413, 1952) and with asymmetric
C'-glycerol from biological sources (SCHAMBYE,
Woop anv Popack. J. Biol. Chem. 206: 875, 1954);
Swick AND NaKAo (ibid 206: 883, 1954). The latter,
in the terminology employed here, was L-glycerol-
1-C. The results conform to established stereo-
chemical relationships and give further support
to the Ogston hypothesis. Information on meta-

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

285

bolic pathways of C!*-glycerol, unobtainable using
the racemate, may emerge using the pure enantio-
morphic forms.

929. Enzymatic degradation of 8-ketoadipic
acid. Masayuki Katagrri* AnD Osamu Haya-
1sHt. Natl. Insts. of Health, Bethesda, Md.
8-Ketoadipic acid (BKAA) has been reported as

an end product formed by cell-free extracts in the

microbial degradation of various aromatic com-
pounds such as phenol, benzoic acid, mandelic
acid, tyrosine, phenylalanine and tryptophan.

This report deals with the further degradation of

BKAA by extracts prepared from tryptophan-

adapted cells of Pseudomonas sp. In the presence

of a catalytic amount of succinyl-CoA and a

substrate quantity of CoA, @KAA was stoi-

chiometrically converted to succinic acid and
acetyl-CoA. When CoA was omitted and a sub-
strate amount of succinyl-CoA was added to the
reaction mixture, the absorption at 305 my in-
creased due to the formation of BKAA-CoA. This
absorption disappeared when either CoA was
added to cleave BKAA-CoA or when succinate was
added to reverse the reaction. The above evidence
suggests the following mechanism for the enzy-
matic degradation of BKAA: 1) BKAA-CoA is
produced by transfer of CoA from succinyl-CoA;

2) BKAA-CoA is then split in the presence of CoA

and thiolase to produce acetyl-CoA and succinyl-

CoA; 8) succinyl-CoA is in turn utilized to activate

BKAA by a cyclic mechanism liberating free

succinate as an end product. Acetoacetate was not

metabolized by this preparation at an appreciable
rate. The fact that the above activity can be dem-
onstrated in extracts obtained from tryptophan
grown cells but not from glucose grown cells
indicates that the enzyme system involved in
these reactions is adaptive and suggests that

BKAA is an intermediate in the microbial degrada-

tion of the above-mentioned aromatic compounds.

930. Effect of insulin on metabolism of C'*
acetate by rat kidney and liver slices.
JoserH Katz aNp JEAN Wona.* Dept. of Physi-
ology, Univ. of California, Berkeley
The metabolism of acetate by rat kidney slices

was investigated using a radioautographic-

chromatographic technique. Qualitatively the
acetate metabolism of kidneys resembles that of
liver. Acetate is oxidized rapidly, incorporated

mainly into glutamate and glucose, and to a

smaller extent into alanine and an unidentified

compound. Smaller amounts are also incorporated
into the intermediates of the Krebs Cycle. Alloxan
diabetes and insulin injection do not materially
affect acetate oxidation, the total acetate metabo-
lism via the Krebs Cycle or its incorporation into
di- and tri- carboxylic acids in either liver or

286 FEDERATION PROCEEDINGS

kidney. However, insulin affects markedly the
incorporation of acetate into glucose and glu-
tamate in both liver and kidney slices, especially
the latter. Insulin treatment of the animals de-
creases glucose formation and increases that of
glutamate. For the incorporation of 1-C'* acetate
the ratio C™ in glucose/C' in glutamate was
about 2 and 0.5 for kidney slices from alloxan
diabetic and normal or insulinized rats respec-
tively. It is concluded that insulin has no direct
effect upon the operation of the Krebs Cycle in
either liver or kidney.

931. Dimerization of bovine serum albumin
with mercurials in water and in concen-
trated urea. Cyrit M. Kay* anp Joun T.
Epsauu. Biological Lobs., Harvard Univ., Cam-
bridge, Mass.

Bovine serum mercaptalbumin (ASH) contains
one sulfhydryl group per molecule. The velocity
constant (ke) for the formation of the mercury
dimer (ASHgSA) and for its dissociation (k_2)
have been studied by light scattering as functions
of pH, temperature, ionic strength and other
variables; and the ‘apparent equilibrium con-

ASHgSA

(ASH) (ASHgX)

determined. The results are qualitatively similar

to those obtained with human mercaptalbumin.

(EpEe.Hoc# et al., J. Am. Chem. Soc. 75: 5058, 1953).

The velocity constant k: attains a maximum

somewhat acid to the isoelectric point, and falls

steadily with increasing negative net charge on the
protein. In isoelectric solution, at 1% total protein
and 25°C, 85-90% of the bovine protein is in the

form of dimer at equilibrium, as against only 60%

for human albumin. Dimerization was also found

to occur in 8 molar urea, ke for the isoelectric
protein being of the order of 240 liters mole“! min™!
as compared with 60 in water. The energy of
activation for dimerization is 11 Kal mole in
urea; & in water. Dimerization in urea solutions
is readily reversible; the equilibrium constant in
urea is close to that in water. The dissymmetry
for the albumin in concentrated urea is negligible,
although the second virial coefficient B is large
and positive, even for the isoelectric protein,
rather than zero as in the case of water. This is
interpreted as an indication of the nearly isotropic
swelling of albumin in concentrated urea solutions.

stant’ K’ = has also been

932. Structure of flavin peptides from suc-
cinic dehydrogenase. Epna B. KEARNEY,
VincENT Massey* AND Tuomas P. SINGER.
Edsel B. Ford Inst. for Med. Research, Henry
Ford Hosp., Detroit, Mich.

Highly purified beef heart succinic dehy-
drogenase has been digested with a mixture of
trypsin and chymotrypsin, and the resulting

Volume 15

flavin peptides have been purified by column
chromatography and paper chromatography. Five
pure flavin peptides, free of extraneous ninhydrin.
reacting material and representing 80% of the
total flavin, were obtained by developing in 4
variety of chromatographic systems. Thege
flavins were analyzed for amino terminal groups
and for total amino acid composition by reaction
with fluorodinitrobenzene. The 5 flavins were
found to have very similar amino acid composition
and structure, differing from each other by having
different end groups, by lacking a whole peptide
sequence, or by being mononucleotide breakdown
products of the original dinucleotide. One fraction,
representing 29% of the total flavin, containing 3
amino end groups and 28-32 amino acids, was fully
as active as flavin adenine dinucleotide in the
p-amino acid oxidase test. Other fractions, dif-
fering only slightly in composition, showed no
coenzymatic activity. All the fractions contained
either 3 or 4 amino end groups. As cystine and
cysteine are absent from all fractions, it is con-
sidered that some, at least, of these peptide
chains are probably bound by esteratic linkages
to ribityl hydroxyl groups, and hence that such
linkages may well contribute to the binding of the
flavin to the enzyme.

933. Procarboxypeptidase. Patricia J,
KELLER,* ELAINE CoHEN* AND Hans NEURATH,
Dept. of Biochemistry, Univ. of Washington,
Seattle.

The chemical and enzymatic properties of
pancreatic carboxypeptidase are well established.
However, in contrast to the pancreatic enzymes
trypsin and chymotrypsin, which are prepared
directly from their purified precursors, carboxy-
peptidase is isolated after activation of the crude
pancreatic juice. Little is known of the relation-
ship of crystalline carboxypeptidase to its zymo-
gen, procarboxypeptidase. Preliminary to a study
of this system, purification of procarboxypepti-
dase was undertaken. A procedure was developed
starting from an acetone powder of beef pancreas.
By successive ammonium sulfate fractionations
and isoelectric precipitations, a protein of 95%
homogeneity was obtained. The purified protein
was inactive toward synthetic substrates for
carboxypeptidase until incubated with trypsin.
Comparison of the molecular properties of the
purified zymogen with those of crystalline car-
boxypeptidase has revealed significant differences.
Procarboxypeptidase is more negatively charged
than carboxypeptidase under the same conditions
of electrophoresis. In univalent buffers of ionic
strength 0.2, the isoelectric point of procarboxy-
peptidase is below pH 4.5, whereas carboxy-
peptidase is isoelectric at pH 6.0. The sedimenta-
tion constant of procarboxypeptidase is 5.87 S as

lume 15

column
Ly. Five
hydrin-
of the
ng in a

These
groups
eaction
1S were
position
"having
peptide
akdown
raction,
rining 3
‘as fully
in the
ns, dif-
wed no
ntained
ine and
is con-
peptide
inkages
at such
g of the

A «
;URATH,
ington,

ties of
lished,
nzymes
repared
arboxy-
e crude
slation-
} zymo-
a study
ypepti-
veloped
ncreas.
nations
of 95%
protein
tes for
rypsin.
of the
ne car-
rences.
harged
ditions
f ionic
irbhoxy-
iboxy-
menta-
87 S as

eae tam

March 1956

compared, with 3.07 S for crystaliine carboxy-
peptidase: Light scattering measurements (per-
formed by J. Kraut) indicate a molecular weight
of 96,000 for procarboxypeptidase, a value almost
3 times that for carboxypeptidase (34,300).
Correspondingly, the specific activity of the
zymogen (proteolytic coefficient after activation)
is about one-third that of carboxypeptidase. Upon
activation with trypsin, procarboxypeptidase can
give rise to a molecule with the sedimentation and
electrophoretic properties of crystalline carboxy-
peptidase. Experiments are in progress to de-
termine the sequence of events involved in the
conversion of procarboxypeptidase to typical
carboxypeptidase.

934. Participation of microsomes in aerobic
pyrophosphate formation. FrRancis_ T.
KENNEY* AND Srpney P. Cotowick. McCollum-
Pratt Inst., Johns Hopkins Univ., Baltimore, Md.
Inorganic pyrophosphate is formed from ortho-

phosphate in respiring liver homogenates (Corr

et al. Biochim. et Biophys. Acta. 7: 304, 1951) but
not in washed mitochondria (LEHNINGER AND

SmitH. J. Biol. Chem. 181: 415, 1949). We have

confirmed these observations in rat liver prepara-

tions oxidizing glutamate in the presence of
magnesium, phosphate, fluoride and AMP. When
mitochondria representing 0.5 gm liver are supple-
mented with microsomes from an equivalent
amount of tissue, the ability to form pyrophos-
phate aerobically is restored: The ratio of pyro-
phosphate formed to oxygen consumed in the
mitochondria-microsome combination is roughly
equivalent to that in the crude homogenate. When
the nuclear fraction or the supernatant fraction is
added to mitochondria, there is no pyrophosphate
formation. Kornberg’s enzyme (J. Biol. Chem.

181: 779, 1950) which forms pyrophosphate and

DPN from ATP and nicotinamide mononucleotide

has been reported to be located essentially ex-

clusively in the nuclear fraction (HoGEBOOM AND

ScHNEIDER. J. Biol. Chem. 197: 611, 1952) and

therefore would not be expected to be concerned

with aerobic pyrophosphate formation. The role
of the microsomes in the latter process is being
studied further,

935. Diphosphopyridine nucleotidase and its
inhibitor from Mycobacterium butyricum.
M. Kern (introduced by W. D. McEtroy).
McCollum-Pratt Inst., Johns Hopkins Univ.,
Baltimore, Md.

A diphosphopyridine nucleotidase (DPNase)
from Mycobacterium butyricum which cleaves the
nicotinamide-ribose linkage of DPN has been
partially purified and characterized. The enzyme
is inactive in crude extracts because of the
presence of an inhibitor. Active preparations can

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

287

be obtained by boiling or acid treatment since
under these conditions the enzyme is stable while
the inhibitor is not. A similar enzyme-inhibitor
relationship has been described for the case of a
DPN pyrophosphatase in Proteus vulgaris by
Swartz etal. (Science, in press). The concentration
of inhibitor required to produce 50% inhibition of
the M. butyricum DPNase was directly propor-
tional to the enzyme concentration, indicating a
relatively low dissociation constant for the
enzyme-inhibitor complex. The system is similar
in this respect to that known for trypsin and its
protein inhibitors. In crude extracts, the inhibitor
was in approximately 10-fold excess over the
concentration of enzyme obtained after boiling.
The DPNase inhibitor is non-dialyzable, precipi-
table with ammonium sulfate, and inactivated by
trypsin, suggesting a protein nature. The inhibitor
has at least a quantitative specificity because at
the concentrations used it has no effect on the
DPNase from pig brain, Neurospora crassa, and
Chromobacterium violaceum.

936. Competition for action of mutarotase by
aldoses exhibiting toxicity. ALBERT S.
Keston. New York Univ. College of Medicine,
New York City.

Under conditions of the mutarotase assay
(Keston. Federation Proc. 14: 234, 1955) 1% levels
of the mutarotase substrates p-galactose, p-xylose,
L-arabinose (all at mutarotational equilibrium)
inhibited the action of 0.4 units of mutarotase on
0.84% glucose by 80%, 50%, and 80%, respec-
tively. L-arabinose when fed in the present study
produced cataract and failure of growth in wean-
ling rats. The diets and conditions were similar
to those which Darby and Day (J. Biol. Chem.
133: 503, 1940) used to produce cataract, hyper-
glycemia, and initial failure of growth with
p-galactose and p-xylose. Keston has proposed
involvement of mutarotase in transport of sugars
and insulin action (Science 120: 355, 1954). Since
competing substrates when present would share
the action of mutarotase (or insulin), it seems
likely that they can inhibit glucose utilization in
organisms. Such inhibitory effects would be
especially important in tissues or organisms which
cannot utilize such substrates (competing with
glucose) at a high rate (e.g. galactosemia) or in
situations where glucose metabolism is already
impaired (e.g. diabetes mellitus). Other points of
competition may be with intermediate compounds
in the glycolytic pathways (e.g. glucose-6-phos-
phate) which possess structural configurations
required for mutarotase action, if mutarotase be
indeed involved in their metabolism. (Inci-
dentally, sugars, not mutarotase substrates, might
show slight effects due to insulin if their metabolic
pathways involve intermediates which are mutaro-

288

tase substrates.) In summary, the sugars known to
be cataractogenic are identical with the competi-
tive substrates of mutarotase or insulin. It is
suggested that such competitive substrates play
a role in cataract formation and other complica-
tions of diabetes mellitus. Galactose, the most
common of these competitive substances in human
diets, may be important in these respects.

937. Inhibition of cholinesterases by alkyl-
phosphates and their reactivation in vivo.
Hetmvut Kewitz (introduced by I. B. Wrison).
Dept. of Neurology, College of Physicians and
Surgeons, Columbia Univ., New York City.

On the basis of the mechanism of cholinesterase
inhibition by certain phosphate containing com-
pounds, I. B. Wilson developed several enzyme
reactivating compounds. Among the most power-
ful up to date is 2-pyridine aldoxime methiodide
(2-PAM). This has been recently shown to be an
antidote against the lethal action of paraoxone in
mice. Therefore, it was of interest to study the
mode of action of paraoxone on cholinesterase
activity in central and peripheral parts of the
nervous system in vivo. Techniques were de-
veloped to measure the extent of enzyme inhi-
bition following the administration of lethal and
sublethal doses of paraoxone, and the effect of
2-PAM on cholinesterase activity was tested.
Using different substrates and concentrations the
types of esterase in the tissues studied have been
determined. The results of these investigations
and their meaning will be discussed. They give
some new aspects to the importance of cholin-
esterase activity.

938. Experimental allergic encephalomyelitic
activity in a glycoprotein fraction of bovine
spinal cord. Marian W. Kies, ELizaBetH
Roxsoz* anp ExviswortH C. Atvorp.* Nail.
Inst. of Mental Health, Bethesda, Md., George-
towy Univ. Med. School, Washington, D. C., and
Baylor Univ. Med. School, Houston, Texas.
Experimental allergic encephalomyelitis is a

disease caused by the injection of whole brain or

spinal cord preparations or active fractions there-
from. Injected animals become paralyzed and
develop characteristic lesions of the brain and
spinal cord. Recently, several investigators have
reported that this activity resides predominantly
in a proteolipide fraction prepared according to

Folch and Lees (J. Biol. Chem. 191: 807, 1951).

Contrary to these findings we have obtained a

highly active preparation in which lipid cannot

be detected. The active fraction has been charac-
terized as glycoprotein consisting mainly of pro-
tein with a small proportion of non-dialyzable
carbohydrate. It was obtained from a bovine
spinal cord preparation which had been partially

FEDERATION PROCEEDINGS

Volume i§

defatted and dehydrated by extraction with ace.
tone and benzene. This preparation was moder.
ately active when an emulsion containing 50 mg
was injected in the adult male guinea pig. The
partially defatted material was then exhaustively
extracted with various organic solvents, and 125
mg of the lipid-free residue found to be active,
The non-lipid residue was extracted with water
under 15 lb. pressure at 120°C for 10 hr. The
water-soluble material was more active at 0.2 mg
dosage than was 50 mg of the cord preparation
used as the starting material.

939. Sequence studies on crystalline papain,
J. R. Kimmet* anp Emit L. Smita. Lab. for
Study of Hereditary and Metabolic Disorders,
Univ. of Utah, Salt Lake City.

Crystalline mercuripapain has been oxidized
with performic acid and digested exhaustively
with crystalline trypsin. The resulting peptide
mixture was chromatographed on a 150 cm column
of Dowex-50X2 with an elution system of increas.
ing pH and ionic strength. There were 21 fractions
identified by reaction with ninhydrin. Amino
acid analysis of those peptides which were judged
to be pure on the basis of amino acid composition
and of end group analysis by the DNP method
account for most of the amino acids of papain,
The first peptide to emerge from the column
(fraction 1) contains 27 amino acid residues, in-
cluding 2 residues of cysteic acid. Since this
peptide lacks lysine or arginine, it is believed to
represent the C-terminal sequence of the enzyme.
Active papain reacts with and is completely
inhibited by one equivalent of iodoacetamide
(Finkle and Smith, unpublished). When this
inactivated papain is oxidized with performic
acid, digested with trypsin and the digest chro-
matographed as described, fraction 1 disappears
from its usual position in the elution diagram,
and a new peak appears. This finding suggests
that iodoacetamide reacts with a sulfhydryl
group at the active site, which must be near the
C-terminal end of the enzyme. Further support
for this concept resides in observations that
mercuripapain can be extensively degraded from
the N-terminal end of the molecule by leucine
aminopeptidase without loss of proteinase ac-
tivity (Hitu anp SmitH. Biochem. et Biophys.
Acta, in press).

940. Solubilization and cytochrome(s) of the
particulates from Acetobacter suboxydans.
Tsoo E. Kine aNp VERNON H. CHELDELIN.
Dept. of Chemistry and Science Research Inst.,
Oregon State College, Corvallis.

Two fractions are obtained from extracting
disintegrated cells of A. suborydans with aqueous
solutions, namely, the supernatant soluble frac-

rec

rat
co
fre
de
37!

plume I§

ith ace.

moder.
g 50 mg
vig. The
ustively
and 125
active,
h water
hr. The
| 0.2 mg

aration

oa pain,
Lab. for
'sorders,

»xidized
istively
peptide
column
increas-
‘actions
Amino
judged
sition
method
papain.
column
ies, in-
ce this
eved to
nzyme,
pletely
tamide
n this
rformic
t chro-
ippears
agram,
uggests
‘hydryl
ear the
upport
s that
d from
leucine
se ac:
Lophys.

of the
ydans.
DELIN.

Inst.,

acting
yueous
» frac-

March 1956

tion and the insoluble residue or particulate frac-
tion. The particulate enzymes from A. suboxydans
were prepared by centrifuging a sonically disin-
tegrated cell preparation in 0.01 m phosphate
buffer at pH 6.0 for 120 min. at 22,000 X g. The
upper, pink layer of the residue was removed
and washed twice by centrifuging in phosphate,
and once in 0.01 m glycylglycine buffer at px 8.0.
The residue was retained for use in 0.01 m glycyl-
glycine buffer. The suspension was treated with
1% deoxycholic acid in glycylglycine buffer, px
8.0 for 5 min. The light scattering of the suspen-
sion was immediately decreased by the treatment.
The mixture was then centrifuged for 150 min. at
an average speed of 25,000 X g in a Spinco prep-
arative centrifuge. A clear yellow extract was
obtained. The protein recovery in the solubilized
extract was about 60%. The extract contained
eytochrome(s). The absorption maxima of the
reduced form were found at 426, 528 and 558 my.
Among them, the peak at 426 my was much the
strongest. Smith (Bact. Rev. 18: 106, 1954) re-
ported the absorption peaks in intact cells of this
organism by a different method at 422, 525 and
554 m. The extract also catalyzed the oxidation
of glucose by molecular oxygen. The oxidation
stopped after one atom of oxygen per molecule
of the substrate.

9441. A column chromatographic and paper
ionographic method for separation of
purines and pyrimidine nucleotides in PNA
hydrolysates. Davin S. Kinnory, Exinor M.
ZoRN AND Dorortuy S. Garngs (introduced by
Atma Hiner). VA Hosp., Hines, and Dept. of
Biochemistry, Stritch School of Medicine, Loyola
Univ., Chicago, Til.

Smith and Markham (Biochem. J. 46: 33, 1950)
determined that treatment of PNA with 1 n HCl
at 100° for 1 hr. resulted in essentially quantita-
tive hydrolysis into the component purine bases
and pyrimidine nucleotides. A column chromato-
graphic and a paper ionographic method for the
separation of these hydrolysis products is here
reported. A multiple column chromatography
system was used which consisted of 6 in-series
connected, progressively longer columns, all 1 cm
in diameter, and ranging, in 1 g. increments,
from 4.5 g. to 9.5 g. of Whatman cellulose powder.
This arrangement accelerated the eluent flow
rate to that through the longest component
column alone and prevented any column segment
from running dry. A gradient solvent dispenser
described by Kinnory et al. (J. Biol. Chem. 212:
379, 1955) was employed. The mixing vessel con-
tained 500 ml of a mixture of 85% tert.-butanol,
3.4% concentrated hydrochloric acid, and 11.6%
water (v/v/v) and the solution in the reservoir
consisted of 70% tert.-butanol, 6.7% concentrated

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

289

hydrochloric acid, and 238.3% water (v/v/v).
Paper ionography of the PNA hydrolysate was
carried out on a strip of S&S 589 green label
filter paper sandwiched between parafilm-lined
glass plates under pressure. Electromigratiop was
carried out in monochloroacetic acid buffer, pH
3.1, ionic strength 0.05, at 9 volts/cm for 6 hr.,
and the components were located by making
contact prints in ultraviolet light. Both methods
achieved complete separation and the chromato-
graphic procedure gave good quantitative re-
coveries.

942. Mechanism of formaldehyde incorpora-
tion into serine. Roy L. Kistiuxk (introduced
by Warwick Saxamr). Dept. of Biochemistry,
Western Reserve Univ. Med. School, Cleveland,
Ohio.

Results previously reported (KisLiuK AND
Saxami. J. Biol. Chem. 214: 47, 1955) demon-
strate that tetrahydrofolic acid is required for
formaldehyde incorporation into serine in Dowex-
1-chloride treated, dialyzed pigeon liver extracts.
In the present studies it is shown that tetrahy-
drofolic acid binds formaldehyde rapidly in the
absence of enzyme and that the bound formalde-
hyde is directly converted to serine-beta-carbon
when enzyme and glycine are added. The rates of
formaldehyde binding and subsequent incorpora-
tion into serine are sufficient to account for the
rate of serine formation from free formaldehyde.
In the presence of a large pool of unlabeled form-
aldehyde, bound C'* formaldehyde is converted
undiluted to the serine-beta-carbon, demonstrat-
ing that formaldehyde is not released from the
tetrahydrofolic acid prior to its condensation
with glycine. C' formaldehyde bound to tetra-
hydrofolic acid is not incorporated into the free
formaldehyde pool in the presence or absence of
enzyme. Paper chromatography of a mixture of
formaldehyde and tetrahydrofolic acid reveals
that at least 2 formaldehyde -tetrahydrofolic
acid derivatives are formed. Folic acid binds no
formaldehyde under the same conditions. This
work provides further evidence that a hydroxy-
methyl derivative of tetrahydrofolic acid is the
active one carbon unit involved in serine biosyn-
thesis.

943. Carbohydrate metabolic pathways in
Acetobacter suboxydans. Paut A. KrTos,*
Tsoo E. Kine anp VERNON H. CHELDELIN.
Science Research Inst., Oregon State College,
Corvallis.

Carbohydrate metabolism in Acetobacter sub-
oxydans has been studied using uniformly labeled
and specifically labeled C™ glucose. Both the
radiochemical recovery of C'4O2 and the oxygen
consumption were 50% of theory for complete

290

combustion in a 150-min. period. The rapid and
successive elimination of C-1 and C-2, together
with the lower yield of C-6, indicates the im-
portance of the pentose cycle in glucose oxidation
in this organism. In addition, a significant elimi-
nation of C-3 and C-4 suggests the presence of at
least one other pathway, possibly glycolysis.
When CH;C“OCOOH was the substrate, over
99% was decarboxylated and converted to ace-
tate, with the remainder reflecting a possible
reduction of pyruvate to triose and further me-
tabolism via the pentose cycle. When 5 uc of
CH;C“OOH were administered to 1 gm. of cells,
less than 0.1% of the C'* appeared as CO:, even
in the presence of (unlabeled) glucose. About 5%
of the activity was incorporated into the cells.
This confirms earlier chemical data that the
Krebs cycle plays little or no role in A. subozy-
dans. Incorporation of administered C'!*O, has
been observed during growth on glucose and yeast
extract. Paper chromatography of an acid hy-
drolysate of the cells indicates labeling in aspartic
and glutamic acids and possibly arginine, while
the medium contains four radioactive compo-
nents, two of which do not appear to be amino

acids.

944. Dissociation of the D-amino acid
oxidase-benzoate complex as a function of
substrate. J. RayMonp KEIN. Biology Dept.,
Brookhaven Natl. Lab., Upton, N. Y.
Inhibition of p-amino acid oxidase by benzoate

has been attributed to competition between

benzoate and substrate for the enzyme. The
usual concept of competitive inhibition indicates
no dependence of the apparent dissociation con-
stant of the enzyme-inhibitor complex upon the
substrate used in the estimation of the constant.

However, conventional estimation of the constant

for the benzoate-oxidase complex gave values

that depend upon the amino acid used. With
valine, oleucine, and phenylalanine, the values

were about twice and, with methionine, about 3

times that obtained with alanine. These findings

are compatible with the possibility that the
enzyme preparation used was a mixture of oxi-
dases each specific for a particular amino acid.

Another possibility is that benzoate reacts re-

versibly with the enzyme-substrate complex to

give the enzyme-inhibitor complex and substrate.

Usual test does not distinguish between the latter

possibility and conventional competitive in-

hibition. (Supported by the Atomic. Energy

Commission.)

945. DNA synthesis in spleens of scorbutic
guinea pigs. Peter D. KLEIN AND RoBert W.
Swick (introduced by Jack ScnHuBERT). Div. of
Biological and Med. Research, Argonne Natl.
Lab., Lemont, Til.

FEDERATION PROCEEDINGS

Volume 1§

Studies have been made of the incorporation of
P*2 into the spleen acid-soluble, RNA and DNA
phosphate of normal and scorbutic guinea pigs,
The specific activities and amounts of these frag.
tions have been measured in normal resting con-
ditions and following a period of hypoxic stimula-
tion. No difference in the specific activities of the
nucleic acids was found when the normal spleens
were compared with the early (14-day) scorbutie
spleens under resting conditions. After the im-
position of a hypoxic regimen the normal spleens
showed a marked increase in specific activity in
both nucleic acids which was paralleled in the
scorbutic spleen only in the RNA. Confirmatory
evidence was obtained in a study of the amounts
of these fractions present in the 4 situations,
The scorbutic spleens were initially higher in
DNA but failed to increase in content during
hypoxia, while the normal spleen doubled its
content of this fraction. (Performed under the
auspices of the Atomic Energy Commission.)

946. Enzymes of nucleoside metabolism in
Escherichia coli. A. L. Koc (introduced by
T. B. Cooper). Div. of Biological and Med.
Research, Argonne Natl. Lab., Lemont, and
Dept. of Biochemistry, Univ. of Chicago, Chicago,
Til.

A single enzyme extract of EZ. coli was tested
for nucleoside splitting enzymes. Ribosides and
deoxyribosides were used as substrates, and the
reaction was measured in various types of buffer
(with and without arsenate) at various pH values
and in the presence of 26 purine and pyrimidine
compounds. From this body of data, the existence
of 2 new enzymes was inferred. First, a hydrolase
acting on inosine, and second a transribosidase
exchanging the hypoxanthine of inosine with
adenine, guanine, xanthine, thymine, 4,5 dia-
minouracil, 5 bromouracil, and 4 (6) aminouracil.
The existence of these enzymes as distinct from
the nucleoside phosphorylases was further estab-
lished by starch block electrophoresis. (Per-
formed under the auspices of the Atomic Energy
Commission.)

947. Formation of serine from glycerol-l,3-
C** by the rat. Roger E. Korrrr, Martin L,
MINTHORN AND Rosert J. Hiwu (introduced
by Joun L. Woop). Div. of Chemistry, Univ. of
Tennessee, Memphis.

The carbon chain of pyruvate is not directly
converted to serine by the rat, since administra-
tion of pyruvate-3-C™ gave serine having ap-
proximately equal radioactivity in carbons 2 and
3 (Nyc AND ZaBin. J. Biol. Chem. 215: 35, 1958).
Another glycolytic intermediate could be a serine
precursor, however, if it is assumed that carbons
2 and 3 of pyruvate are randomized prior to its
conversion to the glycolytic trioses (KREBS.

alt

lume 15

tion of
1 DNA
a pigs,
e frac.
1g con-
‘imula-
| of the
spleens
rbutie
he im-
spleens
vity in
in the
natory
nounts
ations,
her in
during
ed its
er the
n.)

sm in
ced by
1 Med.
t, and
hicago,

tested
28 and
id the
buffer
values
nidine
stence
rolase
sidase

with
> dia-
uracil.
; from
estab-
(Per-
jnergy

1-1,3-
rIn L,
duced
viv. of

rectly
istra-
g ap-
2 and
1955).
serine
rbons
to its
REBS.

March 1956

Bull. Johns..Hopkins Hosp. 95: 19, 1954). To in-
vestigate this possibility, glycerol-1,3-C' has
been administered intraperitoneally to rats.
After death 3 hr. later carcass serine was isolated
and degraded. Carbon 3 of the isolated serine
was found to have 5 times the radioactivity of
carbon 2. Therefore, during the conversion of
glycerol to serine, randomization of carbons 2
and 3 occurred to an extent of only about 33%.
Positions 1 and 2 in liver glycogen were ran-
domized 19%. These data strongly suggest a
3-carbon intermediate of glycolysis (other than
pyruvate) as a precursor of rat serine. The label-
ing patterns found in isolated glutamate, as-
partate and alanine will be presented, as well as
anew degradation of serine proceeding as follows:
serine — alanine — acetaldehyde phenylhy-
drazone — sodium acetate — methyl amine +
CO:. (Supported by the Atomic Energy Commis-
sion.)

48. Alterations in fibrinolytic and coagula-
tion systems of dogs in peptone shock.
SamMuEL N. Kotmen, M. Mason Guest anv D.
R. CELANDER (introduced by ANDREW A.
OrmsBy). Depts. of Physiology and Biochemistry,
Univ. of Texas Med. Branch, Galveston.

Blood of dogs receiving intravenous injections
of Witte’s peptone after exclusion of abdominal
viscera and lower extremities by Nolf’s technic
has been analyzed for fibrinogen, profibrinolysin,
antifibrinolysin, and prothrombin. In addition,
tests for direct fibrinolytic activity of whole
blood by several techniques, of diluted plasma in
a system containing a constant amount of fi-
brinogen, and Lee-White clotting times have
been performed. Studies were made on samples
drawn from the anterior animal before and after
peptone administration (300-800 mg/kg) and from
the posterior animal before and after reconstitu-
tion of the circulation following peptone. Origi-
nally, Nolf observed fibrinolytic activity and
decreased coagulability. These findings are con-
firmed; fibrinolytic activity having been ob-
served in both whole blood and diluted plasma
from the anterior but not from the posterior
animal. Lee-White times in the anterior and
posterior reconstituted animal were significantly
lengthened. Profibrinolysin was not uniformly
altered but antifibrinolysin was decreased in all
post-peptone anterior animal blood samples. In
cases in which fibrinolytic activity developed, the
two stage prothrombin titer was reduced. Ad-
ministration via intravenous drip of 10% glucose
to the anterior animal did not itself produce
fibrinolytic activity but augmented the effects
achieved with peptone. In the presence of glucose,
fibrinogen and prothrombin titers decreased by as
much as 50%. The alterations in prothrombin,
fibrinogen and antifibrinolysin thus appear to he

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

291

the direct result of activation of the fibrinolytic
enzyme system.

949. Relation between platelet metabolism
and platelet integrity. J. L. Koppent anp
Joun H. Otwin (introduced by Dove.as A.
MacFapyEn). Dept. of Surgery, Presbyterian
Hosp. of Chicago, Chicago, Ill.

Incubation of human platelets with enzyme
inhibitors known to affect different aspects of
cell metabolism results in an acceleration of
platelet breakdown. The rate of this breakdown
is dependent upon the nature and concentration
of the inhibitor and is usually greater where a
group of several rather than one specific enzyme
alone is affected. Among the inhibitors capable of
bringing about this effect are substances known
to a) react with sulfhydryl groups, 6) inhibit
dehydrogenase systems, c) inhibit the cytochrome
system, d) interfere with transmethylation and
e) inhibit purine metabolism. That the acceler-
ated rate of platelet breakdown is actually caused
by a disruption of their normal metabolism is
indicated by the fact that it can often be pre-
vented or slowed down in the presence of suitable
metabolite intermediates. It is known that the
integrity of the cell membrane is dependent upon
an actively functioning energy metabolism and
it is suggested that the disruption of metabolism
brought about by the inhibitors results in a de-
crease in the production of available energy. The
apparently close relationship between active
metabolism on one hand and maintenance of
cellular integrity on the other suggests that en-
vironmental conditions affecting platelet metabo-
lism may, even before actual lysis of the platelets,
alter their membrane permeability in such a way
as to bring about an increased rate of release of
intracellular materials. (Supported by the Medi-
cal Research and Development Board, Office of
the Surgeon General, Dept. of the Army, under
contract No. DA-49-007-MD-275.)

950. Polydesoxyribonucleotide synthesis by
enzymes from Escherichia coli. ARTHUR
Kornsera, I. R. LenmMan* anp E. S. Srums.*
Dept. of Microbiology, Washington Univ. School
of Medicine, St. Louis, Mo.

To define the chemical events in the develop-
ment of a bacterial virus, we have explored the
pathways of polydesoxyribonucleotide synthesis
in normal and infected cells. The use of thymidine
was suggested by the report of Friedkin et al.
(Federation Proc. 13: 214, 1954) that C'4-thymidine
is incorporated into the DNA of crude suspen-
sions of chick embryonic tissue. Our studies
started with the observation that 2-C'-thymidine
(generously given us by Dr. M. Friedkin) was
converted by enzyme fractions from normal £.
coli to a polydesoxyribonucleotide and three or

292

more acid-soluble nucleotides. The acid-insoluble
product is made acid-soluble upon treatment
with crystalline pancreatic desoxyribonuclease.
Available evidence suggests the sequence of reac-

I II
tions: thymidine ——> thymidine-5’-P (T5P) ——>

thymidine triphosphate (TTP) III [thymidylate
—

Xx] ane polydesoxyribonucleotide. An enzyme
purified 30-fold from a crude fraction (A) forms
T5P from thymidine + ATP (I). Another enzyme
purified from fraction A forms TTP from T5P +
ATP (II). The conversion of TTP to polynu-
cleotide requires ATP and heat-labile elements
in two discrete, crude fractions (A and B), and
suggests the formation of a nucleotide inter-
mediate (III, IV). The over-all conversion of
C'*-thymidine to polynucleotide requires ATP
and fractions A + B; it is reduced over 50% by
an equimolar amount of unlabeled T5P but not
by higher levels of desoxyadenylate, desoxy-
guanylate and desoxycytidylate. P*?-T5P con-
version to polynucleotide also requires ATP and
fractions A + B; it is inhibited by thymidine
polyphosphates synthesized by the Khorana
procedure. Rates of conversion of thymidine,
T5P and TTP (1 X 10-° m) are, respectively,
0.3, 0.5 and 1.0 um/mg protein/hr. In T2-phage
infected cells, these reactions have also been
observed, but at a much diminished rate.

951. Further properties of partially purified
pancreatic cholesterol esterase. M1tcHELL
Korzenovsky, B. M. VEesELy aNp ERo.p R.
DILLER (introduced by O. K. Benrens). Lilly
Research Labs., Eli Lilly and Company, In-
dianapolis, Ind.

Synthesis and hydrolysis of cholesterol esters
catalyzed by partially purified pancreatic choles-
terol esters catalyzed by partially purified pan-
creatic cholesterol esterase have been studied
with the manometric procedure of Korzenovsky,
Rust and Diller (Federation Proc. 12: 772, 1955).
The enzyme exhibits a specific requirement for
cholic acid both for synthesis and hydrolysis of
esters such as cholesteryl oleate. The optimum
concentration of cholic acid is the same for all
substrates studied. Conjugates of cholic acid are
inactive in the system as are all other common
bile acids, either free or conjugated. In addition
to the cholic acid requirement, the in vitro system
must contain an appropriate concentration of
electrolyte for maximum activity. The. stimula-
tory effect of the electrolyte is not a property of a
particular cation or anion, but appears to be a
function of the concentration of cations or anions.
Ammonium sulfate is as effective as sodium chlo-
ride is the latter is used in twice the concentra-
tion.

FEDERATION PROCEEDINGS

Volume if

952. Release of bacteriophage DNA from its
protein covering. Luoyp M. Koztorr. Depi,
of Biochemistry, Univ. of Chicago, Chicago, I,
The distal half of the tail protein of bacterio.

phage T. can be removed by the specific action
of complexes of the zinc group metals (Kozuorp
AND HENDERSON. Nature 176: 1169, 1955). During
viral invasion a similar alteration of the viral
tail protein occurs and then the viral DNA is
injected into the host cell. The effect of metal
complexes on protein components and the factor
controlling the release of viral DNA from the
shortened phage have been investigated. It wag
found that complexes such as Cd(CN)s;~ break
the disulfide bonds of cystine and oxidized gluta-
thione. Cysteine and reduced glutathione were
identified among the reaction products. The
reaction occurs under conditions (25°, pH 7.7)
comparable to those in which T, is inactivated,
The release of viral DNA from Cd(CN)s3~ treated
T2 does not occur immediately after alteration of
the tail structure but depends upon the px and
the nature of buffer. With pu 8.75 tris(hydroxy-
methyl) aminomethane (Tris) buffer 80-98% of
the viral DNA is released from its protein cover-
ing within 3-5 min. at 37°. Below px 8.0 there is
little or no release of viral DNA. However, px
8.75 ammonium and borate buffers release only
10-20% of the viral DNA even after prolonged
incubation. Since Tris has an amino group ad-
jacent to two hydroxyl groups the effect of glu-
cosamine at pH 8.75 was tested. Glucosamine,
which is a normal constituent of the host cell
wall, also caused the release of viral DNA from
its protein covering.

953. Formation of oxalacetate from d-
tartrate. L.O. KRAMpPITZ AND FEoDOR LyNEN.*
Inst. ftir Zellchemie an der Deutschen Forschung-
sanstalt fiir Psychiatrie (Max Planck Inst.)
Miinchen, Germany.

A gram positive bacillus was isolated by soil
enrichment which anaerobically dissimilated d-
tartaric acid according to the following equation:
d-tartrate — 0.19 lactate + 0.85 acetate + 1.3
COz + 0.45 He. The organism was cultivated ina
medium consisting of inorganic salts, d-tartrate
and ammonium sulfate. We have been most in-
terested in the initial reactions of the dissimila-
tion, particularly the possibility that d-tartrate
(or some derivative) may be dehydrated to enol
oxalacetate. Resting cells of the organism rapidly
decarboxylated oxalacetate with the formation
of hydrogen. Equimolar concentrations of hy-
droxylamine totally inhibited the production of
carbon dioxide and hydrogen from d-tartrate
with a concomitant disappearance of hydroxyla-
mine. Spectral evidence indicated that the pro-
duct formed was the oxime of oxalacetate. An

olume 15

rom its
F. Dep,
ago, Ill.
racterio-
c action
K OZLOFP
. During
he viral
DNA is
of metal
> factors
rom the
. It wag
~ break
d gluta-
ne were
ts. The
pH 7.7)
tivated,
treated
ation of
PH and
ydroxy-
98% of
1 cover-
there is
ver, pH
se only
olonged
yup ad-
of glu-
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ost cell
A from

m d-
‘YNEN,*
schung-

Inst.)

by soil
ted d-
uation:
+ 1.7%
ed ina
artrate
ost in-
simila-
artrate
(0 enol
-apidly
mation
of hy-
‘ion of
urtrate
‘oxyla-
e pro-
te. An

March 1956

extract of the organism prepared by grinding with
alumina rapidly dissimilated d-tartrate with the
formation of carbon dioxide and hydrogen.
Hydroxylamine also inhibited this activity with
a disappearance of the amine. Heat treatment of
the extract in a 70° bath in a manner that the
extract attained a temperature of 64° in 75 sec.
(considerable denaturing of protein) resulted in
a preparation which no longer decarboxylated
oxalacetate much above the rate of spontaneous
decarboxylation. The heated extract formed
only small quantities of carbon dioxide from d-
tartrate, however evidence for oxalacetate forma-
tion was obtained by analysis of the reaction
mixture with 6 ketonic acid reagents. The un-
treated extract contained malic dehydrogenase
which was inactivated by the heating procedure.
The formation of oxalacetate from d-tartrate by
the heated extract was coupled with malic de-
hydrogenase and reduced diphosphopyridine
nucleotide with the formation of d malic acid.

954. Rate of reduction of various substrates
by the hydrogenase system. ALVIN I. KRAsNA
AND D. Rirrensera. Dept. of Biochemistry,
College of Physicians and Surgeons, Columbia
Univ., New York City.

The activity of the enzyme hydrogenase is
generally assayed by determining the rate of re-
duction of some substrate with molecular hy-
drogen. The substrates so employed include such
diverse compounds as methylene blue, the y,7’
dipyrydyls, nitrate, sulfate and oxygen. If hy-
drogenase is the enzyme which reversibly ac-
tivates molecular hydrogen it is reasonable to
expect that other enzyme systems or co-factors
may be involved in the transfer of electrons orig-
inating in the hydrogen to the reducible sub-
strate. The catalysis of the exchange reaction
between hydrogen and heavy water and the
ortho-para conversion of hydrogen are properties
of the enzyme which do not require other factors
or hydrogen acceptors. We have studied the
hydrogenase of Desulfovibrio desulfuricans, Pro-
teus vulgaris, E. coli, Rhodospirilum rubrum and
Clostridium pasteurianum. In these organisms
there is no obvious relationship between the rate
of the exchange reaction and the rate of reduction
of methylene blue or benzyl viologen or the rate
of evolution of hydrogen from reduced benzyl
viologen. The differing rates of these reactions
are probably a consequence of the different
mechanisms involved in the interaction between
the activated enzyme, i.e., the enzyme hydride
(KRASNA AND RiITTENBERG, J. Am. Chem. Soc. 76:
3015, 1954) and the reducible substrate. Only the
exchange reaction measures the rate of reaction
of hydrogenase with molecular hydrogen. Methyl-
ene blue was found to greatly increase the lag

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

293

phase of the exchange reaction in Proteus vulgaris.
This was shown to be due to the inhibition of
the oxygen removing systems by methylene blue.

955. Provitamin A activity of beta-carotene-
free nonsaponifiable material from alfalfa.
R. F. Krauss. Dept. of Biochemistry, School of
Medicine, West Virginia Univ., Morgantown.
One hundred mg of nonsaponifiable material

were extracted from approximately 100 gm of

fresh Range variety alfalfa leaf which was labeled
with C™ as a result of growing the plants in an
atmosphere of C4O.. The specific activity of this
nonsaponifiable material was 11,000 CPM/mg.

From a petroleum ether solution of this material

55 mg of Xanthophyll material were removed by

adsorption on tricalcium phosphate. The specific

activity of this material was 14,500 CPM/mg

(Fraction I). Approximately 100 ug of pure beta-

carotene were isolated from the remaining petro-

leum ether solution but it had no radioactivity.

The remaining nonsaponifiable material amounted

to 43 mg and had a specific activity of 5,100 CPM/

mg (Fraction II). Equal parts of Fraction I

were fed to 2 rats. At the end of 24 hr. they were

killed, livers pooled and vitamin A isolated which
had a specific activity of 100 CPM/mg. Similar
feeding and treatment of Fraction II resulted in
producing liver vitamin A with a specific activity
of 40 CPM/mg. The vitamin A content of each
liver was approximately 1000 ug/gm of liver. In
each animal less than 5% of the radioactivity fed
was recovered in the feces. (Aided by grant from
Natl. Vitamin Fndn., Inc.)

956. Phosphorylase b to phosphorylase a
reaction. Epwin G. Kress anp Epmonp H.
FiscuEr. Dept. of Biochemistry, Univ. of Wash-
ington, Seattle.

The conversion of phosphorylase 6 to phos-
phorylase a, observed previously in crude ex-
tracts of rabbit skeletal muscle (FiscHER AND
Kress, J. Biol. Chem. 216: 121, 1955), has been
studied as an isolated system with purified com-
ponents. Requirements for the reaction include
Mn++ or Mg* ions, ATP, and an enzyme desig-
nated temporarily as the b to a converting en-
zyme. This enzyme has been purified 65-fold and
is free of PR enzyme activity. Experiments with
P%2 labeled ATP have shown that there is in-
corporation of the isotope into phosphorylase a
formed in the b to a reaction. The phosphate is
present in a form that is not released on precipita-
tion of the protein with trichloroacetic acid, but
is released when phosphorylase a is converted to
phosphorylase b by the PR enzyme. In the puri-
fied phosphorylase 6 to a converting system, no
effect of epinephrine or glucagon has been ob-
served.

294

957. Mechanism of in vitro hydroxylation.
Rosert C. KrueGer (introduced by F. F.
Heyrrotu). Dept. of Biological Chemistry, Univ.
of Cincinnati College of Medicine, Cincinnati,
Ohio.

A model aerobic hydroxylating system con-
sisting of ethylene diamine tetraacetic acid
(EDTA), ascorbic acid (AH:), and an iron salt
has lately come under study (UDENFRIEND et al.,
J. Biol. Chem. 208: 731, 1954; DatetiesH, Arch.
Biochem. 58: 214, 1955). The present report is an
attempt to demonstrate that this system has
characteristics similar to the well-known ferrous
ion (Fe**)-hydrogen peroxide (H2O2) system, the
Fenton reaction, and to the ‘redox activation’
systems used in various ‘cold’ rubber recipes.
Hydroxylations and polymerizations are carried
out in these latter systems by virtue of the free
hydroxyl] radicals produced. Assuming formation
of H.O. by the autoxidation of AHs, the system
of Udenfriend and the ‘redox activation’ systems
have the following in common: 1) a peroxide, 2) a
reducing agent, 3) a chelating agent, and 4) a
metal catalyst. It has been demonstrated that
AH: can reduce the Fe***-EDTA complex, a fact
which lends support to the possibility of a redox
reaction operating in the system of Udenfriend.
Free hydroxyl radicals produced by ionizing
radiations (Hart, J. Am. Chem. Soc. 73: 68, 1951)
and, as shown in the present report, by the reac-
tion between Fe** and H.0, will decarboxylate
formic acid. In similar fashion, the EDTA—
AH,—Fe** system under aerobic conditions will
decarboxylate formate. These results in conjunc-
tion with information in the literature support
the thesis that the EDTA—AH.—Fe* system
functions by way of free hydroxyl radicals.

958. Enzymatic components of the tartrate
oxidizing system of beef heart mitochon-
dria. Ernest Kun anp Davin D. Daviss.*
Inst. pr Enzyme Research, Univ. of Wisconsin,
Madison.

A soluble enzyme system prepared from beef
heart mitochondria (J. Biol. Chem. 218: No. 1,
1956) catalyzes two DPN-linked dehydrogena-
tions: 1) tartrate + DPN+ = oxaloglycolate +
DPNH + H?; 2) oxaloglycolate + DPNt+ =
diketosuccinate + DPNH + Ht. Decarboxyla-
tion of oxaloglycolate to hydroxypyruvate is
catalyzed by Mg** while the formation of gly-
oxylate from oxaloglycolate occurs in the presence
of Mg** + versene. Both enzymic reactions are
determined by measuring the reoxidation of
DPNH by the appropriate substrate in phosphate
buffer (0.5 M, px 6.5) containing versene. The
substrate used in reaction 1 is the stable enol
form of oxaloglycolic acid (dihydroxyfumaric
acid), which is converted to an unstable keto

FEDERATION PROCEEDINGS

Volume 1§

form by Mg** + versene. Purification by negative
adsorption on charcoal, gradient elution of
(NH,)2SO, precipitates and chromatography will
be reported. Preparations of specific activity of at
least 30,000 for reaction 1 and 18,000 for reaction 8
have been obtained. The unit of enzyme activity
is defined as a change in absorbance at 340 my of
0.01/min. in a total volume of 1 ml with a 1-em
light path.

959. Muscle and liver cytochrome oxidase
activity in rats and in three species of large
animals. H. O. KunKE.. Dept. of Biochemistry
and Nutrition, Texas A. and M. College System,
College Station.

Calculations of total liver, kidney and brain
cytochrome oxidase activity in mice, rats and
dogs and of the cytochrome oxidase activities per
unit weight of rat skeletal and adult rat and dog
cardiac muscle have suggested a quantitative
relationship of this enzyme system to basal meta-
bolic rates (J. Biol. Chem. 198: 229, 1952). Using
rats and 3 species of large animals, sheep, swine
and cattle, the cytochrome oxidase activities of
the gracilis muscle and of the liver were deter-
mined and compared with the body weight. The
muscle cytochrome oxidase activity was found to
be roughly an inverse function of the body mass.
Statistical evaluation after converting all values
for weight and enzyme activity to logarithms
resulted in a regression equation of log A =
2.318 — 0.239 (log W) where log A is the logarithm
of the muscle cytochrome oxidase activity (ul
O2/mg/hr.) and log W the logarithm of the body
weight in kg (r = 0.89). The cytochrome oxidase
activity per unit weight of the liver does not
reflect the variation of body mass in the sheep,
swine and cattle series. However, statistical
evaluation yielded a regression of the logarithm
of total liver cytochrome oxidase activity (l
O:/liver/hr.) of 0.674 (r = 0.98) on the logarithm
of body weight. These regression coefficients are
of the order of magnitude expected from the {
power rule of relationship of basal heat produc-
tion of body weight and the assumption of a
direct linear relationship between cytochrome
oxidase activity to the basal metabolic rate.
From these and other data it is suggested that
cytochrome oxidase activity acts as a sensitive
indicator of the organismic level of metabolism.

960. Action of fluoride on bone salts. K.
KuTNERIAN* AND A. C. Kuyper. Dept. of
Physiological Chemistry, Wayne Univ. College of
Medicine, Detroit, Mich.

Precipitates of bone salts were formed under
physiological conditions of temperature and p#
from solutions which contained calcium and phos-
phate in slight excess of concentrations normally

pre
cal
pre
the
for
cr

dey
pre
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in |
on
of

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cre
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cal
cor

961

— tw we 6

of
ear
iso

pat
cor
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wel
hay
fec
cor
ato
rad
ine
Ea
div
the

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egative
ion of
hy will
ty of at
action g
ctivity
D mu of
a l-cm

»xidase
f large
emistry
System,

1 brain
ts and
ties per
nd dog
‘itative
| meta-
, Using
, Swine
ities of
deter-
it. The
und to
r mass,
values
rithms
; As
arithm
ity Gl
e body
»xidase
es not
sheep,
tistical
arithm
ity (ul
arithm
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the }
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:s. K.
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under
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| phos-
rmally

March 1956

present in blood, and physiological levels of bi-
carbonate, magnesium, and sodium chloride. The
presence of fluoride (0.5-12 parts per million) in
these precipitating solutions decreases the rate of
formation of the precipitate and appears to in-
er ase its solubility. This effect of fluoride is
dependent upon the presence of magnesium. On
prolonged equilibration, fluoride decreases the
solubility of the precipitate. Iodide and bromide
in levels up to 50 parts per million have no effect
on the rate of precipitation. When a small amount
of fluoride is added to a system of bone salt in
equilibrium with a supernatant solution the
fluoride is incorporated into the precipitate. This
is accompanied by a decrease in the solubility of
the precipitate and an increased exchange with
radioactive calcium thus suggesting that fluoride
may enter the precipitate not by a process of
simple exchange with hydroxyl ion, but by dis-
solution and reformation of a new precipitate.
Under similar conditions fluoride causes no in-
crease in the exchange of calcium with coarsely-
powdered teeth (60 mesh); however, with finely-
powdered teeth (200 mesh) the incorporation of
fluoride is accompanied by increased exchange of
calcium. Except when used in relatively high
concentrations fluoride has no influence on the
carbonate content of bone salts.

961. Radioyttrium with complex heterogene-
ous carrier. GRANVIL C. Kykmr, JOHN Rar-
TER,* Encar A. CrEss* AND NELSON STEVENS.*
Med. Div., Oak Ridge Inst. of Nuclear Studies,
Oak Ridge, Tenn.

Previously reported studies illustrate the effect
of the size of dose of certain individual rare-
earth elements on the mobilization of their radio-
isotopes. In other combinations the effect of the
dose of one of these elements on the radioisotopic
pattern of another was evaluated. The various
combinations gave quite similar patterns when
parenteral doses ranging from 107 to 10-4 m/kg
were administered to rats and mice. These studies
have been extended to include the combined ef-
fect of mixed rare earths. A commercial product
composed largely of the lighter lanthanons (96%,
atomic numbers 57-60) was used as carrier for
radioyttrium. Intraperitoneal studies in mice
include doses of 10-", 10-8, 10-7, 10-*, 10-*, and
10‘ m of rare earth per kg at 1, 5, and 10 days.
Each animal in the groups of 6 was analyzed in-
dividually. The fractions of the dose remaining in
the excreta were determined. Both the rate and
degree of mobilization were reduced by increased
doses of the mixture.

962. Use of ioresin column for the determina-
tion of PBI-131 conversion ratios. T. N.
Laur AND D. L. TaBERN (introduced by FLtoyp

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

295

MclInt1RE). Abbott Labs., North Chicago, IIl.

Previous attempts to use ion exchange resins
for the separation of I-131 and thyroxine bound
activity in plasma samples have involved either
centrifugation of the resin or the use of long glass
columns. Through the use of a more active and
finely divided anion exchange resin, formed into
the column at the time of use, it has been found
possible to use a column only 2-3 cm long. The 2
or 5 ce plasma sample is counted first for iodide
plus thyroxine activities. It is then poured over
the column, the time required for passage of the
plasma and two saline washes being 6-8 min.
Removal of iodide is better than 99%. The ef-
fluent, likewise assayed in a well type scintillation
counter is a quantitative measure of the protein
bound thyroxine. No separations or centrifuga-
tions are required. Conversion ratio = cts. in
effluent/cts. in original sample. An all plastic
system, assembled from standard parts, is so
inexpensive, that at the conclusion of the test it
is discarded intact, thus eliminating the possi-
bility of errors due to cross contamination.

963. Inhibition of sugar utilization by nys-
tatin. J. O. Lampren, Exvutiotr R. Morean*
AND ADELAIDE C. Stocum.* Squibb Inst. for
Meéd. Research, New Brunswick, N. J.

Dr. Harlyn O. Halvorson has observed that the
antifungal agent nystatin inhibits the aerobic
and anaerobic utilization of glucose and maltose
by yeast (personal communication). Inhibition
occurred only after a lag period. In our studies 3
ug of nystatin produced half maximal inhibition
of the utilization of glucose, maltose, or fructose
by 2.5 mg (dry weight) of yeast. When excess
nystatin was present glucose remained in a form
utilizable by glucose oxidase. Endogenous oxygen
uptake was also eliminated. The lag is short at
pH 4.5, prolonged at pH 6.8 and was not reduced
by preincubation of nystatin in the succinate-
phosphate buffer (pH 4.5). Increasing the nys-
tatin:cell ratio shortened lag time. Organisms
whose growth is inhibited by nystatin (C. al-
bicans, S. cerevisiae, P. notatum) had sensitive
sugar-utilizing systems, whereas glucose utiliza-
tion by E. coli or S. fecalis (whose growth is not
inhibited) was not affected by 100 ug of nystatin
per ml. Actively glycolyzing cell-free extracts
were prepared from Fleischmann’s fresh bakers
yeast. With these preparations CO production
from glucose was not affected by 200 ug nystatin
per ml nor was hexokinase activity reduced. These
extracts, when inactivated by ageing, did not
prevent nystatin from inhibiting glycolysis by
intact cells. Normal amounts of hexokinase were
present in extracts prepared from cells which had
been preincubated with nystatin until glucose
utilization ceased. The inhibition of sugar utiliza-

296

tion does not then appear to result from an in-
hibition of hexokinase but may represent an
effect on sugar transport or ATP regeneration.

964. Dissociation of purified prothrombin.
Francots Lamy (introduced by Kart Scumip).
Dept. of Biology, Massachusetts Inst. of Tech-
nology, Cambridge, Mass.

Purified prothrombin was kindly furnished by
Dr. Walter Seegers (Record Chem. Progress,
winter issue, 1952). It behaves as a monodispersed
system in phosphate buffer at pH 7.0 and ionic
strength 0.15. Its molecular weight (Physiol.
Rev. 34: 722, 1954) is 68,000. However, this ‘mole-
cule’ is a complex which yields sub-units whose
characteristics depend upon the nature of the
solvent and whether or not thrombin is evolved.
In phthalate buffer px 4.92-6.0 and ionic strength
0.15, sub-units of m 34,000 appear which are in
equilibrium only with a quadrimer. A similar dis-
sociation is observed in phosphate buffer in the
ultracentrifuge at prothrombin concentrations
below 1 mg/ml. In either case thrombin is not
produced. Prothrombin has been dissolved in
25% sodium citrate and studied after various
lengths of time. The sedimentation and diffusion
coefficients were found to have constant values
of Sao® = 2.86 and Da® = 6.23 X 107 cm?/sec.
This corresponds to molecular weight of 37,000
and axial ratio of 7.3 if the molecule is assumed
to be a prolate ellipsoid of revolution. On diluting
a 10% solution of prothrombin in 25% citrate, a
series of additional fragmentations and aggrega-
tions takes place (loc. cit.). Thus, the only ob-
served physical change in concentrated citrate is
a splitting of prothrombin into two sub-units
and a loosening of intramolecular structure.

965. Function of vitamin A. M. Danret LANzE,*
GrorGE Wo.r* anp B. Connor JOHNSON.
Div. of Animal Nutrition and Radiocarbon
Lab.,%Univ. of Illinois, Urbana.

In order to obtain a clue to the function of
vitamin A (outside of vision), an attempt was
made to detect a biochemical lesion in rats made
vitamin A deficient, by comparing their metabo-
lism of radioactive acetate-1-C'* (9.17 mc/mm)
with that of pair-fed and ad libitum fed control
animals. No effect of the deficiency was found on
expired carbon dioxide and liver protein labeling.
Specific and total activities in cholesterol and
fatty acids were higher in the deficient than in
the controls. It was concluded that vitamin A is
not required in their biosynthesis. However, the
ratio of specific activities of fatty acids/choles-
terol revealed a depression for the deficient
animal. These results suggest a decreased biosyn-
thesis of fatty acids compared to cholesterol in
the deficient animal. To study glycogen forma-

FEDERATION PROCEEDINGS

Volume 15

tion, each rat was fed one gram of glucose (un.
labeled) prior to injection of the labeled acetate.
A pronounced lowering of total glycogen content
and radioactivity compared to the pair-fed con-
trol was found in the deficient animal, suggesting
an involvement of vitamin A in glycogen bio.
synthesis.

966. Human placental estradiol-178 dehy.
drogenase. LorNA LANGER* AND Lewis |,
Eneeu. Med. Labs., Collis P. Huntington Me-
mortal Hosp. of Harvard Univ., at Massachusetis
General Hosp., Boston.

Estradiol-178 dehydrogenase catalyzes the re-
versible dehydrogenation of estradiol-176 to
estrone, requiring DPN as hydrogen acceptor,
The enzyme has been partially purified from
human term placenta by means of ammonium
sulfate precipitation and calcium phosphate gel
adsorption. A®-36-hydroxysteroid dehydrogenase
activity present in the initial extracts is removed
by this purification. The instability of enzyme
preparations is lessened by lyophilization, or by
the presence of 50% glycerol, estradiol-178, or
DPN. Enzyme activity is assayed by measuring
the formation of DPNH spectrophotometrically,
and of estrone and other steroids chromatographi-
cally. Cysteine is added to enzyme preparations
to obtain maximal activity, suggesting the pres-
ence of an essential sulfhydryl group. Estradiol-
178 dehydrogenation is markedly inhibited by
10-5 m p-chloromercuribenzoate. This inhibition is
largely reversed by cysteine. In addition, estra-
diol-178 or DPN protects the enzyme to some
extent from inhibition by p-chloromercuribenzo-
ate. The reaction rate varies little with pH in the
range 6.0-9.7, but there is a peak in enzyme ac-
tivity at pH 9.7-10.3. The enzyme shows high
specificity for estradiol-178. Estradiol-17a is not
dehydrogenated, nor is estriol. The activity of
the enzyme toward neutral 178-hydroxysteroids
is markedly less than toward estradiol-176, under
conditions optimal for estradiol-178 dehydrogena-
tion.

967. Porphyrin biosynthesis in nucleated
avian erythrocyte. Earn G. LARSEN* AND
JAMES M. OrtTEN. Dept. of Physiological Chemis-
try, Wayne Univ. College of Medicine, Detroit,
Mich.

Practicable quantities (10-20 ml) of intact
chicken erythrocytes following incubation with
glycine and acetate under aerobic conditions for
18-24 hr. did not contain sufficient quantities of
porphyrins to permit definitive studies of the in-
dividual porphyrins. However, the addition of
adenosine (1 mg/ml packed cells) together with
added glycine and acetate and incubation under
the same conditions, increased the porphyrin

sti
fr¢

lume 15

ose (un-
acetate.
content
ed con-
sgesting
en. bio.

dehy.
wis L,
ton Me-
chuselis

the re-
178 to
ceptor,
d from
nonium
ate gel
genase
emoved
enzyme
, or by
178, or
asuring
rically,
graphi-
rations
e pres-
Tadiol-
ted by
ition is
_ estra-
> some
ibenzo-
in the
me ac-
s high
is not
rity of
teroids
under
ogena-

leated
* AND
‘hemis-
etroit,

intact
1 with
ns for
ties of
he in-
ion of
r with
under
yhyrin

March 1956

content ofthe cells 3-5-fold. These conditions
were employed in subsequent studies to deter-
mine the effect of various substances on porphyrin
biosynthesis. Sedormid decreased all porphyrin
fractions except the uroporphyrin fraction.
Isonicotinic acid hydrazide (1 mg/ml packed
cells) markedly decreased the total porphyrin
content of the cells. Preliminary studies indicate
that 3-acetimide-5-methyl tetronic acid signifi-
cantly increased the porphyrin content of the
cells. The possible effect of 2,5- and 5,6-dimethyl
and 5,6-dichloro-benzimidazoles are also being
studied. (Supported by Grant No. C-2144(c)
from the Natl. Insts. of Health.)

968. Effect of glutamate on coupled phos-
phorylation in brain. E. C. Layne, JR.*
AND SAMUEL P. BessmMan. Dept. of Pediatrics,
Univ. of Maryland School of Medicine, Balti-
more.

The addition of 0.01 m glutamate to dialyzed
rat brain homogenates respiring in succinate and
fortified with ATP and DPN leads to a 10-fold
increase over the control in oxygen consumption
and a 5-fold increase in alpha-ketoglutarate,
with no increase in citrate, ammonia, or gluta-
mine formation. These preparations, which have
little ability to oxidize fumarate or malate, show
increased substrate disappearance and _ keto-
glutarate formation when glutamate is added.
This effect, which also can be demonstrated
anaerobically, is shown to be caused by a trans-
amination of glutamate with oxaloacetate, since
fumarate disappearance can be accounted for
completely by malate, oxaloacetate (as pyruvate)
and aspartate formation. The glutamate utilized
can be accounted for by the ketoglutarate formed.
The individual oxidation of glutamate, succinate,
fumarate or malate is accompanied, in our prep-
arations, by a coupled phosphorylation, but the
addition of glutamate to malate, fumarate, or
succinate leads to a consistent uncoupling. Since
the concentration of glutamate in brain is 0.01
M, these results suggest that glutamate might
exert a significant effect on Krebs cycle oxidation
and phosphorylation in brain. (Aided by grants
from Natl. Science Fndn. and Playtex Park
Fndn.)

99. Relationship of propionate and suc-
cinate in the Propionibacterium. F. W.
LEAVER AND R. StJERNHOLM (introduced by
J. H. Jones). Univ. of Pennsylvania School of
Veterinary Medicine, Philadelphia, and Western
Reserve Univ. School of Medicine, Cleveland,
Ohio.

We have allowed propionate-1,3-C' (20.8 and

26.5 counts per minute per micromole, c/m/um

respectively) to be metabolized by resting cells of

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

297

Propionibacterium arabinosum. The reisolated
propionate was 100% randomnized in the 2-3
carbons. The carboxyl carbon had 19.6 c/m/um
and the ethylene carbons 11.2 c/m/um. The ratio
of activities of the carboxyl to ethylene carbon
was 1.75. The ratio in the succinate was 1.18;
hence it was evident that succinate was not re-
sponsible for the randomization of tracer in the
propionate. Otherwise, one would expect that
the ratio of activities in the propionate and suc-
cinate would be identical. In another experiment
propionate-1,3-C'* was allowed to be metabolized
for 2-5 min. by a large mass of resting cells. The
external acids were isolated and degraded. The
cells were then refluxed for 3 hr. in 0.5 N sulfuric
acid and ether extracted. This fraction was
labeled ‘internal’ acids. Degradation of the
‘internal’ malate, succinate and propionate in-
dicated that all 3 had comparable specific activi-
ties and that the ratio of activities of their car-
boxy] to ethylene carbons were similar suggesting
that they were all in equilibrium. However, there
was a marked difference in the ratio of specific
activities between the internal and external
acids. Hence, it would appear that there are 2
pools of propionate and succinate and that pro-
pionate and succinate may be formed by more
than one pathway.

970. Mammalian gluconokinase. Irwin G.
LEDER (introduced by Mary E. Maver).
Natl. Insts. of Health, Bethesda, Md.

The studies of Stetten and Stetten (J. Biol.
Chem. 187: 241, 1950) indicated that gluconate
was converted extensively to glucose and to COz
in the rat. Since less than 1% of the glucose de-
rived from gluconate is formed by direct reduc-
tion (STETTEN AND Topper, J. Biol. Chem. 203:
653, 1953), the most probable pathway for glu-
conate metabolism is via phosphorylation to 6-
phosphogluconate. Extracts of hog kidney have
been found to catalyze the phosphorylation of
gluconate in the presence of Mg.{ and ATP. The
enzyme, gluconokinase, has been purified ap-
proximately 100-fold and its properties have
been studied. The product is active as substrate
for 6-phosphogluconic dehydrogenase and _ its
identity as 6-phosphogluconate has been con-
firmed by paper chromatography in formic acid-
methanol and ammoniacal methanol solvent
mixtures. The pH optimum, established in maleate
buffer, is 6.2. The partially purified enzyme is
relatively stable at 2°C, but heating at 52°C for
8 min. destroys over 95% of the activity. Several
metals can substitute for Mg.t. In decreasing
order of activity these are Mnt, Znt, Cof, and
Cat; the last being 3 as active as Mgt. The en-
zyme preparation contains highly active ade-
nylate kinase but is free of ATPase activity. By

298

means of the lactic dehydrogenase-phosphoenol-
pyruvate kinase system, the specificity of glu-
conokinase has been studied bv testing for the
appearance of ADP in the presence of various
possible substrates. Of the following compounds
tested, only p-gluconate is active: p-gluconate,
p-glucose, p-glycerate, p-gulonate, p-glucuronate,
p-altronate, p-galactonate, D-arabonate, L-ara-
bonate and L-gluconate.

971. Purification of adenylic-5’-phosphoric
acid deaminase. Ya-pIN LEE (introduced by
Marvin J. JoHNSON). Inst. for Enzyme Re-
search, Univ. of Wisconsin, Madison.
Adenylic-5’-phosphoric acid deaminase of

rabbit muscle, first reported by Schmidt, has been

purified about 200-fold over the initial extract.

This enzyme is specific, deaminating only ade-

nylic-5’-phosphoric acid (AMP), and its activity

can be observed by a decrease in optical density
at 265 my as reported by Kalckar. In our experi-
ments enzyme activity was measured by noting
the time for decrease in optical density at 265 mz

from 0.55 to 0.40 in a solution containing 0.1 m

succinate buffer pH 6.4 and 5 X 10-° m AMP.

Rabbit skeletal muscle was extracted with 0.3 m

KCl-0.15 m potassium phosphate buffer, px 6.5,

and the low salt-insoluble fraction was separated

by diluting 10-fold with water. The activity could
be separated from the precipitate by heat treat-
ment as reported by Engelhardt and co-workers.

The filtrate obtained after heat treatment was

fractionated between 7.4 and 23 vol. % of 95%

alcohol and dissolved in 0.5 m KCl. The am-

monium sulfate cut between 0.30 and 0.45 satura-
tion was dissolved in 0.5 m KCl and dialyzed
against 0.02 m succinate buffer. The precipitate
contained 15-25% of the activity of the original
extract. Under the above test conditions 8.5 y of
the purified preparation in 3-ml solution caused
an optical density change from 0.55 to 0.40 in
about 2#sec. The maximum activity of the puri-
fied preparation in 0.1 m succinate buffer and

5 X 10-° m AMP was in the range pu 6.3-6.6. In

the presence of 0.5 m KCl and pu 7, the activity

is quite stable at 0°C for at least 2 wk.

972. 3-4 Dihydroxyphenylethylamine as a
precursor of adrenal epinephrine in the
intact rat. LemMuEL C. LEEPER* AND SIDNEY
UDENFRIEND. Lab. of Chemical Pharmacology,
Natl. Heart Inst., Natl. Insts. of Health, Be-
thesda, Md.

In previous reports from this laboratory it has
been shown that C' labelled phenylalanine,
tyrosine and 3-4 dihydroxyphenylalanine (DOPA)
can serve as precursors of adrenal epinephrine in
the intact rat. In this report evidence will be
presented that 3-4 dihydroxyphenylethylamine-

FEDERATION PROCEEDINGS

Volume 1§

1-C* (DOPAmine) can also be converted to
adrenal epinephrine. When 7,500,000 c.p.m. of C¥
DOPAmine were administered to rats according
to the procedure of Udenfriend and Wyngaarden
(Biochim. and biophys. acta—in press) the spe-
cific activity of the isolated adrenal epinephrine
was found to be about 10,000-20,000 c.p.m/um,
Since these values are 5-10 times greater than
those found when C'* DOPA was used it indicates
that DOPAmine is a more immediate precursor
of epinephrine and suggests that DOPA acts
as a precursor following decarboxylation to
DOPAmine.

973. Simple procedure for the isolation of
brain sulfatides. MARJoRIE LEES (introduced
by Aurrep Pops). McLean Hosp. Research
Lab., Waverley, Mass.

Brain white matter is extracted with chloro-
form:methanol and the extract washed with
water as described by Folch, Lees and Sloane-
Stanley (Federation Proc. 13: 209, 1954). The
washed extract is mixed with petroleum ether,
methanol, and water, 1:0.5:0.75:0.2, v/v. The
system separates into 2 phases; the upper one ig
mixed with eth its volume of water, and the
small lower phase that separates is discarded.
The remaining upper phase is mixed with N
aqueous KCl, methanol and water, 1:0.04:0.02:
0.1 v/v. The resulting lower phase is collected
and mixed with petroleum ether and water, 1:
0.12:0.05 v/v, with enough methanol to maintain
a single phase. The solution is placed at —10°C
and the resulting precipitate collected by filtra-
tion. It contains 3.3% sulfur and after recrystalli-
zation exhibits the chemical composition of Blix’s
cerebron sulfuric acid. The solutes in the filtrate
contain 0.6% sulfur which has been concentrated
into a fraction containing about 1.8% sulfur and
1.8% phosphorus. It may represent a_sulfo-
phosphatide. The sulfatides obtained account
for about half the total lipide sulfur of the tissue.
This yield can be increased by reprocessing the
discarded fractions. The procedure can be run in
2 days. By comparison, the lengthy procedure of
Blix yielded only 3% of total lipide sulfur as
cerebron sulfuric acid. (Supported in part by
Research Grant B-130, Natl. Insts. of Health.)

974. Oxidative phosphorylation with mito-
chondria and particles of beef heart. R. L.
Lester,* D. M. ZiEGLER* AND D. E. GREEN.
Inst. for Enzyme Research, Univ. of Wisconsin,
Madison.

The P/O ratio for each step of the citric acid
cycle carried out by fresh sucrose suspensions
of beef heart mitochondria (I) is about 2 except
for oxidation of succinate (P/O ca. 1). When I is
repeatedly frozen and thawed in various media,

ume 15

ted to
. of Cu
-ording
aarden
ne spe-
»phrine
-n/uM,
r than
dicates
cursor
A acts
ion to

on of
»duced
esearch

shloro-
| with
sloane-
|.
ether,
. The
one ig
nd the
arded,
ith N
t:0.02:
lected
er, 1:
intain
—10°C
filtra-
‘stalli-
Blix’s
iltrate
trated
ir and
sulfo-
count
Lissue.
ig the
run in
ure of
fur as
rt by
alth.)

mito-
R. L.
REEN.
onsin,

e acid
nsions
xcept
n I is
nedia,

March 1956

phosphorylation remains unimpaired except for
the succinic step. DPN is required for full oxida-
tive activity. I is stable for months when stored
at —10°. When suspensions of I are brought to
pu 8.5 three easily separable fractions can be
prepared: 1) heavy particles readily sedimented
(II); 2) light particles sedimented at 110,000 X
g (III); and 3) a particle-free supernatant. IT
shows essentially the same phosphorylation pat-
tern as I except that the phosphorylation accom-
panying succinic oxidation is relatively low and
that various cofactors are now needed for full
oxidative activity (DPN, TPN, CoA, GSH,
cocarboxylase, etc.). When DPNH is used instead
of the substrates of the citric acid cycle no phos-
phorylation attends the vigorous oxidation. III
is the submitochondrial electron transfer particle
(cf. abstract by J. Glenn and F. L. Crane) which
can catalyze the rapid oxidation of succinate or
DPNH and the slow oxidation of a-ketoglutarate.
Under optimum conditions the 2,4-dinitrophenol
sensitive phosphorylation accompanying oxida-
tion of a-ketoglutarate has a P/O ratio of about
1. ATPase is negligible in I, evident in II and
most pronounced in III.

75. Differentiation of various types of car-
bohydrates by means of vanillin in acid
medium. Victor E. LeviNE AND WILLIAM W.
Becker.* Dept. of Biological Chemistry and
Nutrition, Creighton Univ. School of Medicine,
Omaha, Neb. *

Aldopentoses, uronic acids, methylpentoses,
aldohexoses and ketohexoses respond differently
when made to react with an aqueous vanillin
solution in the presence of hydrochloric or sul-
furic acid. Reactions carried out with 1 ml con-
taining 1 mg of carbohydrate solution, 1 ml of
1% vanillin solution and 1 ml of concentrated
sulfuric acid or 3 ml of concentrated hydrochloric
acid yield a purple color with aldopentoses and
uronic acids, a green or blue-green color with
methylpentoses, and a bright red color with
ketohexoses and disaccharides, trisaccharides
and polysaccharides containing ketohexose units.
Aldohexoses, and disaccharides and _ polysac-
charides composed of aldohexose units yield a
yellow color with vanillin and hydrochloric acid.
Vanillin and hydrochloric acid also give a yellow
color. With vanillin and sulfuric acid, however,
aldohexoses carbohydrates yield an orange color.
The reaction mixtures involving aldopentoses,
methylpentoses and ketohexoses produce dis-
tinctive absorption spectra and follow Beer’s
law. With hydrochloric acid and vanillin the
colors on heating tend to fade out within a defi-
nite period of time. Using 1 ml of a mixture con-
taining 1 mg each of glucose, fructose, ribose
and fucose, the yellow color given by glucose

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

299

appears first, but disappears readily to be fol-
lowed by the red color produced by fructose. This
color in turn fades out to be followed by the
purple color yielded by ribose. This color also
disappears to be replaced by the stable green
color typical of fucose. (Aided by a Public Health
Service grant.)

976. y-Glutamyl phosphate. Leon LEvintow
AND ALTON MeErsTER. Natl. Cancer Inst., Natl.
Insts. of Health, Bethesda, Md.

Although no free intermediate has been de-
tected in the course of the enzymatic synthesis
of glutamine (glutamate + ATP + NH;= gluta-
mine + ADP + P), the possibility of an inter-
mediate anhydride between glutamate and phos-
phate is suggested by the results of O'8 transfer
studies (Boyer et al. Federation Proc. 14: 185,
1955; Kowausky et al., Abstracts 127th Meeting
A.C. 8., 27C, 1955). The instability of y-glutamyl]
phosphate has rendered its study difficult in our
hands. Accordingly, N-acetyl-y-u-glutamy] phos-
phate has been synthesized by a procedure anal-
ogous to the preparation of B-aspartyl phosphate
(Buack AND Gray, J. Biol. Chem. 213: 27, 1955).
N-acetyl-y-L-glutamyl phosphate is hydrolyzed
by purified amino acid acylase (BrRNBAUM et al.
J. Biol. Chem. 194: 455, 1952), and the y-u-gluta-
myl phosphate thus formed reacts nonenzymati-
cally with NH,Cl to form t-glutamine, which
has been identified by chromatography and a
specific enzymatic test. The addition of a puri-
fied enzyme preparation which catalyzes the
synthesis of glutamine from glutamate, ATP and
NH;, to a reaction mixture consisting of N-
acetyl-y-L-glutamyl phosphate, NH,Cl and acyl-
ase did not enhance the rate of formation of
glutamine. The addition of Mgt*, ADP, or £-
mercaptoethanol was likewise ineffective. These
results are consistent with the concept that
free y-glutamyl phosphate does not participate
in the enzymatic synthesis of glutamine, but do
not exclude the participation of an enzyme-
bound anhydride of glutamate and phosphate.

977. Conversion of estradiol-178-16-C'* to
radioactive 16-ketoestradiol-178 in man.
Mortimer Levitz, JEAN R. Spitzer aND GRAY
H. Twomsty (introduced by Istpor GREEN-
WALD). Dept. of Obstetrics and Gynecology,
College of Medicine, New York Univ., New York
City.
16-Ketoestradiol-178-16-C!4 (Z) (2.5 uwe/mg) was

synthesized by treating radioactive dimethyl-

marrianolate 3-benzyl ether with sodium in liquid
ammonia. J was administered to 2 human subjects.

About 12% of the urinary radioactivity excreted

in the first 24 hr. was recovered (isotopic dilution

method) as unchanged J, provided hydrolysis was

300 FEDERATION PROCEEDINGS

carried out with 8-glucuronidase. Only 2% was
recovered when the urine was hydrolyzed with
hydrochloric acid. These experiments indicated
that J could be isolated from the urine if it were a
metabolite of estradiol-178-16-C!*. Subjects were
given a single intramuscular injection of estradiol-
17B8-16-C!* (1.5 mg, 6.1 we/mg) with 4.5 mg of un-
labeled 16-ketoestradiol-178 in propylene glycol.
Radioactive 16-ketoestradiol-178 was recovered in
the urine. This was demonstrated by the following
procedure. After 8-glucuronidase hydrolysis and
ether extraction in the presence of 15 mg of carrier,
the metabolite was purified successively by column
chromatography on silica gel, countercurrent
distribution and paper chromatography. The
radioactive 16-ketoestradiol-178 was eluted, di-
luted with 60 mg of additional carrier and crystal-
lized to constant specific activity. The purified
product contained 550 c.p.m/mg. Three derivatives
were prepared. Each possessed the same molar
specific activity as the parent compound. In the
urine samples analyzed, 1.5% of the radioactivity
was contained in 16-ketoestradiol-178.

978. Hydrogen transfer in DPN-linked enzyme
reactions. H. RicHarp LEvy,* JoHN L. GRAvEs*
AND BrrGiIT VENNESLAND. Dept. of Biochemistry,
Univ. of Chicago, Chicago, Ill.

Both glucose dehydrogenase and triosephos-
phate dehydrogenase have previously been shown
to catalyze a direct transfer of hydrogen between
substrate and DPN. The steric specificity of this
transfer with respect to DPN has now been
determined. This was demonstrated with glucose
dehydrogenase by reducing DPN witb 1-deutero-
glucose, and reoxidizing the DPND with pyruvate
in the presence of lactic dehydrogenase. All the D
was retained in the nicotinamide ring of the
oxidized DPN. In another experiment, DPN
containing D in position 4 of the nicotinamide
ring (SAN Pietro, J. Biol. Chem. 217: 579, 1955)
was reduced with unlabeled glucose. When the
DPND so formed was reoxidized enzymatically
with pyruvate, all the D was transferred to the
lactate formed. Similar results were obtained in
an analogous experiment performed with yeast
triosephosphate dehydrogenase. These results
show that both glucose and triosephosphate de-
hydrogenase exhibit B-stereospecificity for DPN,
similar to the B-OH steroid dehydrogenase pre-
viously studied. This means that the H is trans-
ferred from the substrate to the opposite side of
the nicotinamide ring from that utilized in the
alcohol, lactic, and malic dehydrogenase reac-
tions. (The side of the nicotinamide ring utilized
by yeast alcohol dehydrogenase has been desig-
nated @, and the opposite side 8.) All the enzy-
matic, DPN-linked aldehyde and alcohol oxida-
tions hitherto studied have been shown to cause

Volume 1§

direct and sterospecific hydrogen transfer. Unlike
the above classes of enzymes, however, dihydro.
orotic dehydrogenase, in one preliminary experi-
ment, did not cause a direct transfer of hydrogen
from DPNH to dihydroorotic acid.

979. Denaturation of bovine serum albumin,
Mivton Levy AnD Rosert C. WaRNER. Dept. of
Biochemistry, New York Univ. College of Medi-
cine, New York City.

The processes of denaturation lead to a de.
creased solubility of bovine serum albumin (BSA)
as a result of heating. Many variations of px and
salt concentration leave native BSA in solution,
A limited number of these conditions precipitate
heated BSA from solution. We have systematically
studied the solubility of native and heated BSA
in systems of variable salt concentration at con-
stant pH. The salts used were ammonium sulfate
and sodium trichloroacetate. The solubility of
native BSA is a monophasic function of salt
concentration. The solubility of heated (px 7.0,
65.7°, 0.2 ionic strength, phosphate) BSA is a
biphasic function of salt concentration. The less
soluble fraction (P;) changes its properties as the
heating continues. It can be separated from
native and a more soluble P2 fraction by salting
out. The isolated fractions P: and P; retain their
properties of solubility and behavior to further
heating. Therefore irreversible processes led to
their formation. P; is a mixture of polymers which
increases in size and complexity as heating con-
tinues but does not revert to P2. P2 has nearly the
same size and solubilities as native BSA but is
more slowly converted to P; on continued heating.
Intra and inter molecular interchanges of —S—S—
and —SH in the protein are important for the
processes studied. A sulfhydryl blocking agent,
parachloromercuribenzoate greatly reduces the
initial rate of formation of P; whereas addition of
cysteine accelerates the initial rate. After a time
the rates appear to become identical probably then
being dependent on the balance between oxidation
of —SH and hydrolysis of —S—S—.

980. Isolation of androsterone glucuronoside.
Marvin L. LEwBart* AND JOHN J. SCHNEIDER.
Dept. of Medicine, Jefferson Med. College, Phila-
delphia.

Urine obtained following the oral administra-
tion of sodium dehydroisoandrosterone hemisuc-
cinate to a normal man was acidified and extracted
with n-butanol. Paper chromatography of aliquots
of the crude extract using the systems ethyl ace-
tate-n-hexane-10% acetic acid (150:100:250),
n-butyl acetate-n-butanol-10% formic acid
(80:20:100) and ethyl acetate-n-butanol-10%
formic acid (90:10:100) demonstrated the presence
of dehydroisoandrosterone sulfate, three other

plume If

. Unlike
lihydro-

experi-
y drogen

bumin,
Dept. of
f Medi-

> a de-
2 (BSA)
PH and
olution,
cipitate
atically
ed BSA
at con-
sulfate
ility of
of salt
‘PH 7.0,
A is a
“he less
s as the
d from
salting
in their
further
led to
s which
ng con-
urly the

but is
eating.
-s—g—
for the
agent,
es the
ition of
a time
ly then
idation

10side.
/EIDER.
_ Phila-

nistra-
misuc-
tracted
liquots
yl ace-
0: 250),
acid
01-10%
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other

March 1986

17-ketostéroid sulfates and two 17-ketosteroid
glucuronosides. Repeated extraction with ethyl
acetate of a 1% aqueous acetic acid solution of a
portion of the crude extract gave a fraction which
contained most of the glucuronosides and only
small amounts of the sulfates. Countercurrent
distribution of this fraction through 50 transfers
using the system ethyl acetate-n-hexane-1% acetic
acid (150:75:225) gave two incompletely separated
components. The contents of tubes 25 through
34 were combined and redistributed in the same
system. Following the second distribution, the
contents of tubes 24 through 32 were combined to
give what appeared to be a single crystalline
substance. When chromatographed on paper the
recovered glucuronoside was homogeneous and,
following hydrolysis with 6-glucuronidase, andros-
terone, identified by melting point and infrared
spectroscopy, was the only steroid recovered. The
glucuronoside also was characterized as the
crystalline triacetyl methyl] ester.

981. Amino-acid content of carbonic an-
hydrase. Grorce T. Lewis AND LORETTA
LANGEN.* Dept. of Biochemistry, Univ. of Miami
School of Medicine, Coral Gables, Fla.

Carbonic anhydrase was obtained in crystalline
form in 1942 but the content of only 2 amino acids
has been reported. Three highly purified prepara-
tions of the enzyme were available and each of the
3 has been analyzed for its content of 18 amino
acids. Determinations were made microbiologically
using Lactobacillus arabinosus 17-5, Streptococcus R
fecalis or Leuconostoc mesenteroides P-60. Pre-
liminary hydrolysis of the enzyme protein was
carried out in a sealed tube in the presence of HCl
or NaOH. Reliable values were obtained for 9
amino acids—arginine, histidine, methionine,
threonine, leucine, glutamic acid, tyrosine, valine
and tryptophane. Determined with lesser accuracy
were isoleucine, lysine, alanine, phenylalanine
and cystine. The values for proline, aspartic acid,
glycine and serine represented gross approxima-
tions only. The contents of cystine and tyrosine
agreed rather closely with those reported pre-
viously and determined by the use of chemical
procedures. No differences were detectable in the
amino acid content of the 3 preparations analyzed.
(Sponsored, in part, by the Biochemical Research
Dept., J. B. Roerig and Co. (Div. of Charles
Pfizer & Co., Inc.), Chicago, Ill.)

982. Guanosine triphosphate in the conver-
sion of inosinic acid to adenylic acid.
Irving LigBERMAN. Dept. of Microbiology,
Washington Univ. School of Medicine, St. Louis,
Mo.

An enzyme has been partially purified from
extracts of Escherichia coli which catalyzes the

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS 301

synthesis of adenylosuccinate (CARTER AND
Couen, J. Am. Chem. Soc. 77: 499, 1955) from
I5P and L-aspartate in a reaction involving GTP
as follows: I5P + L-aspartate + GTP — adenylo-
succinate + GDP + Pj. GSP, GDP, and the di-
and triphosphates of adenosine, cytidine, uridine,
and inosine could not replace GTP. L-aspartate
could not be replaced by NH;, D-aspartate, or by
any of 6 other related amino acids. The reversi-
bility of the reaction was tested with 0.5 um of
adenylosuccinate, 0.5 um of GDP, and 10 um of
P;**. Anion-exchange chromatography revealed
the presence of 0.062 um of P®? in the GTP area.
When enzyme or adenylosuccinate was omitted,
0.0016 and 0.0033 um of P22 were found in the same
area, respectively. When 6-O-labeled I5P (4.90
atom % excess) was used as a substrate, P; (4.88
atom % excess) but not the acid-labile phos-
phates of GDP and GTP (0.00 atom % excess)
became labeled. These results are consistent with
an hypothesis that 6-phosphoryl-I5P is an inter-
mediate in the synthesis of adenylosuccinate.
Adenylosuccinate was further metabolized by
cell-free extracts to yield A5P, a reaction first
described by Carter and Cohen.

983. Nucleoside metabolism in human erythro-
cytes and components. Fasian J. LIoNETt1,*
Wittram L. McLevian* anp Burnuam S&S.
Waker. Dept. of Biochemisiry, Boston Univ.
School of Medicine, Boston, Mass.

We have demonstrated that erythrocyte ghosts
are able to convert inorganic phosphate to cellular
ester phosphate (Biochim. et biophys. acta 18: 443,
1955). Such cells do not metabolize glucose but
can utilize the pentose moieties of nucleosides as
energy sources. The effects of inosine, guanosine,
and adenosine are now shown to be approximately
the same on the uptake of inorganic phosphate
and disappearance of total pentose in ghosts and
erythrocytes. The amount of phosphate esterified
in the ghosts is somewhat greater than that oc-
curring in erythrocytes but is appreciably less
than that in cell-free hemolysates. The ratios of
the change in organic phosphate to pentose catab-
olized are more dependent upon the initial
concentration of phosphate present than nucleo-
side and approaches unity for the cellular com-
ponents. The activation energies for inosine-
stimulated phosphate exchange are 34,000 cal/M
for erythrocytes, 19,000 cal/M for ghosts, and
9,000 cal/M for the hemolysates. The metabolic
character of phosphate utilization by the ghost is
verified, while variations in activation energies
among the cell components may be indicative of
similar processes characterized mainly by differ-
ences in permeability. Present data are con-
sistent with phosphorolytic cleavage of inosine,
yielding hypoxanthine and ribose phosphate.

302 FEDERATION PROCEEDINGS

The ribose phosphate is further metabolized
yielding ketopentose, triose, and other products.
The purine constituent is apparently not further
involved. (Supported by a grant-in-aid from the
Massachusetts Div. of the American Cancer
Society, and by a contract with the Surgeon
General of the U. 8. Army.)

984. Solvation of DDT by lipoprotein for study
of DDT-dehydrochlorinase. H. Lipke, H.
Crespi, C. W. Kearns Anp B. R. Ray (intro-
duced by R. E. Jonnson). Depts. of Entomology
and Chemistry, Univ. of Illinois, Urbana.

The insolubility of DDT in aqueous media
(0.04 ug/ml) has previously required the use of 2-
phase systems for measurement of DDT-dehydro-
chlorinase (DDT’ase) in extracts of DDT-resistant
houseflies (STERNBURG ef al., J. Agr. and Food
Chem. 2: 1125, 1954). This enzyme shows unusual
sensitivity to the surface active agents commonly
employed in enzymatic studies with lipophilic
substrates, and procedures using emulsions or
DDT-coated beads severely limit the scope of
kinetic studies. The ultracentrifugal flotation
technique of Gofman and co-workers (Methods of
Biochem. Anal. 1: 459, 1954) has been utilized to
obtain large quantities of stable egg yolk lipo-
protein for solvation of DDT and similar water-
insoluble substances. When the lipoprotein is
exposed to DDT following clarification by extrac-
tion with ether, a preparation is obtained con-
taining 400-800 ng DDT/ml of 2% lipoprotein
solution thus permitting the inclusion of DDT in
the enzyme system at substrate levels. Since the
product of DDT’ase action, DDE, absorbs
strongly ia the ultra-violet region, a rapid and
direct spectrophotometric assay for the enzyme
has been devised. Enzyme and lipoprotein-DDT
are added to the Beckman cuvette and the rate of
increase of optical density at 260 mu is measured
following the addition of the glutathione co-factor.
In the pregénce of purified DDT’ase preparations,
the solvating effect of the lipoprotein permits
virtually complete dehydrochlorination of the
substrate to proceed in 40 min.

985. Polyribonucleotide synthesis with an
enzyme from Escherichia coli. Urte. Z.
LitTAvER (introduced by Metvin Conn).
Dept. of Microbiology, Washington Univ. School of
Medicine, St. Louis, Mo.

Studies in this laboratory on the enzymatic
pathways of ribonucleotide synthesis have led to
investigations of mechanisms of polyribonucleo-
tide formation. Adenine-8-C14-labeled ATP, pre-
pared via the condensation of adenine and 5-
phosphoribosylpyrophosphate and incubated with
particulate-free extracts of E. coli, is converted to
an acid-insoluble product. The enzyme purified

Volume 1§

over 60-fold on the basis of this activity reacts
preferentially with ADP, and at a rate 40 times
greater than with ATP. One mg of the purified
preparation polymerizes 200 um of ADP per hr.
at 37°. Over 50% of the ADP (.0016 m to .016 m)
may be converted to the product. The latter,
tentatively identified as a polyadenylate, is
insoluble at px 3.5 and produces a viscous solution
at neutral pH. Uracil-2-C!*-labeled UDP reacts
with the purified enzyme fractions. The formation
of polyadenylate is readily reversed by inorganic
phosphate. Polyadenylate (0.15 um/ml) is split to
the extent of 14%, 29%, and 95% in the presence
of phosphate at respective concentrations of
6.4 X 10° a, 4.4 X 10-4 m, and 1.9 X 10-3 M. The
over-all reaction can be formulated by the equa-
tion: n nucleoside-P-P = (nucleoside-P), + n Pj,
and thus is an example of the reaction described
by Grunberg-Manago and Ochoa for an enzyme
from Azotobacter vinelandii (J. Am. Chem. Soc. 77:
3165, 1955). P*? exchange studies of the reaction
mechanism and an investigation of the synthesis
and breakdown of the polyribonucleotide in-
hibitor of E#. coli desoxyribonuclease are in
progress. (Support for the work was provided by a
Dazian Fndn. Fellowship and grants to Dr. A.
Kornberg from the Public Health Service and the
Natl. Science Fndn.)

986. Cell-free incorporation of C!‘-amino acids
into cytoplasmic ribonucleoprotein par-
ticles. JouHn W. LitrLeFrreLp* AND ELIZABETH
B. Kewier. Med. Labs., Collis P. Huntington
Memorial Hosp. of Harvard Univ., at Massa-
chusetis General Hosp., Boston.

A recent report from this laboratory indicated
that in rat liver the cytoplasmic ribonucleo-
protein particles are the initial site of amino acid
incorporation into protein both in vivo and in
cell-free preparations. Similar particles have now
been isolated from the microsome fraction of
Ehrlich ascites tumor cells by treating a distilled
water lysate with 0.5 m NaCl followed by differ-
ential centrifugation. As in liver the initial in-
corporation of C!‘-amino acids by whole ascites
tumor cells is into these particles. They have a
ribonucleic acid to protein ratio 0° 1.0-1.2, show 2
major ultracentrifugal components (cf. Peter-
mann, Mizen and Hamilton), and are active in the
cell-free system, in contrast to similar particles
isolated from liver by the use of sodium deoxy-
cholate. Thus t-leucine-1-C" or t-valine-1-C" is
incorporated into the particles in the presence of
enzymes precipitated at pu 5 from either liver or
ascites tumor soluble cell proteins, plus 10 um/ml
of crystalline ATP and 0.25 um/ml of guanosine
triphosphate. This system is simi'ar to that from
liver (Zamecnik and Keller) as to rate and dura-
tion of incorporation, inhibition by ribonuclease,

er-
the
les
xy-

se,

AMERICAN SOCIETY OF

March 1956

specificity for L-amino acids, and requirement for
guanosine triphosphate. The incorporation of
amino acids by the isolated ribonucleoprotein
particles with the soluble enzymes demonstrates
that microsomal components other than these
particles are unnecessary. Furthermore a system
to regenerate ATP is no longer required, probably
because the ascites tumor cell fractions show less
ATPase activity than do the liver fractions.

987. Conversion of labeled sugars to L-ascorbic
acid in ripening strawberries. FRANK A.
Lorwvus, Rosir JANG AND C. G. SEEGMILLER
(introduced by E. F. JANSEN). Western Utiliza-
tion Research Branch, Agric. Research Service,
U.S. Dept. of Agriculture, Albany, Calif.
Individual ripening strawberries which had been

removed from the plant were either injected with
or stem-fed microcurie amounts of p-glucose-1-
C4, p-glucose-2-C!*, p-galactose-1-C!*, or Na
p-glucuronate-6-C!4. After 24-120 hr., each berry
was ground up in boiling water and the various
berry acids recovered from the supernatant by
means of gradient elution on an anion exchange
column. L-ascorbie acid (AA) recovered in this
manner represented 70-90% of the total reduced
AA present in the homogenate. AA from glucose-
1-C'4 fed berries had a specific activity roughly
comparable to that of the citric and malic acid
fractions. After dilution with carrier and crystal-
lization to constant specific activity, the reduced
AA was degraded as described by Horowitz and
King (J. Biol. Chem. 125: 200, 1953). The di-o-
amino anil of dehydroascorbic acid was also pre-
pared and degraded. The distribution of activity
in berries fed p-glucose-1-C!* was as follows:
C-1 (65-70%), C-6 (12-20%), and C-3 (5-12%),
and in berries fed p-glucose-2-C!*, C-2 (70%) and
C-5 (20%). This pattern of labeling between
the triose moieties corresponds to that observed
earlier in the hexoses of wheat seedlings by Edel-
man, Ginsburg and Hassid (J. Biol. Chem. 848:
218, 1955) and in the p-galacturonic acid isolated
from the pectin of strawberries fed p-glucose-1-C'4
AA from a p-galactose-1-C!4 fed berry was ap-
proximately equally labeled in C-1 and C-6.
Very little activity was incorporated into the
AA of berries fed Na p-glucuronate-6-C!*. These
results strongly suggest that in the strawberry,
the intact carbon chain of a hexose precursor
closely related to p-glucose is preserved in the
transformation from hexose to AA. Further, there
is no inversion of configuration in the course of
this transformation; that is, C-1 of p-glucose
becomes C-1 of AA.

988. Glycogenesis in the liver in normal and
depancreatized rats. Ropert W. LoNGtey,*
Ouav H. Atvie* anp JosepH H. Roe. Dept. of

BIOLOGICAL CHEMISTS 303
Biochemistry, School of Medicine, George Wash-
ington Univ., Washington, D.C.

Normal and totally depancreatized adult rats
were injected intraperitoneally with 10% solu-
tions of fructose, or glucose, or invert sugar, at a
dosage of 2 gm/kg of body weight after a 24-hr.
fast. Groups of animals were killed at approxi-
mately hourly intervals over a 12-hr. period by
injection of Nembutal. The whole liver was quickly
removed, weighed, and homogenized in 5% tri-
chloroacetic acid solution. The glycogen in the
acid extract was determined by the anthrone
method of Carroll, Longley, and Roe (J. Biol.
Chem., in press). The liver glycogen values ob-
served were maximal around 3 hr. after sugar
injection and did not return to preinjection levels
until about 12 hr. after administration. Fructose
formed liver glycogen most rapidly, glucose gave
the slowest response and invert sugar yielded
values intermediate between those given by
fructose and glucose. The rate of glycogenesis in
the liver after fructose injection was the same in
depancreatized rats as in normal animals. Fol-
lowing glucose injection the rate of formation of
liver glycogen in the totally depancreatized rats
was as rapid as, or slightly more rapid than,
in the normal animals. The data suggest that
pancreatectomy has no effect upon the formation
of liver glycogen from parenterally administered
fructose and glucose.

989. Reduction of corticosteroid secretion in
pantothenic acid deficient rats. BERNARD
B. LoNGWELL, ARNOLD E. ReEtr* AND ELIZABETH
Hanssury.* Dept. of Biochemistry, The Lovelace
Fndn., Albuquerque, N. Mex.

Direct determinations of corticosteroids in
adrenal vein blood of rats confirm previous indi-
cations that adequate levels of pantothenic acid
(P.A.) are essential for normal adrenal function.
150 gm male Wistar rats were fed a) 10 days on a
commercial P.A. deficient diet followed by 6)
approximately 14 days with addition of 0.5 gm cal-
cium w-methylpantothenate/100 gm deficient diet.
Deficiency had developed at this time as evidenced
by hemorrhagic lesions and histologic changes in
the adrenal. Two control groups were pair-fed
to the deficient rats with diets (a) and (b) supple-
mented with calcium pantothenate, respectively,
6.6 and 11.6 mg/100 gm diet. After this feeding
schedule, adrenal vein blood was collected by a
cannulation method and corticosteroids were re-
covered by dialysis against aqueous methanol,
solvent extraction and paper chromatography.
Eluted steroids were quantitated by their optical
density at 240 my as well as by the blue tetrazolium
or the Zimmerman reaction. No difference in cor-
ticosteroid output was observed between the
2 control groups. Actual recoveries of corti-

304 FEDERATION PROCEEDINGS

costerone, the predominant corticosteroid in the
rat, averaged 8.9 ug/ml whole blood and 136
ug/adrenal/kg/hr. for the control groups (10 rats)
and 5.4 ug/ml whole blood and 80 yg/adrenal/
kg/hr. for the deficient group (13 rats). The de-
crease in corticosterone output by P.A. deficient
rats was paralleled by a decrease in other adrenal
cortical steroids.

990. Induction of sexual differentiation in
hydra by a volatile chemical or gas. W. F.
Loomis. The Loomis Lab., Greenwich, Conn.
Hydra possess 2 alternative pathways of cellular

differentiation, i.e. their ectodermal interstitial

cells differentiate at times into well defined
gonads but not at others. Previous workers have
shown that sexual differentiation is not an obliga-
tory phase in the life cycle of hydra (which may
reproduce alternatively by asexual budding), but
rather an epiphenomenon occurring in response
to apparently environmental factors. Although
previous attempts to define the responsible fac-
tors have been unsuccessful, temperature, nutri-
tion, crowding, stagnation and reduced oxygen
tension (W. F. Loomis, Science 120: 3108, 1954)
have been implicated in the process. It has re-
cently been found in this laboratory that hydra
secrete a sex-hormone-like substance into their
culture water that in turn induces them to differ-
entiate sexually. A 10-day assay system for this
compound has been constructed. Present results
indicate that the compound is heat stable, neutral
and highly volatile with a low solubility in water.
It is not oxidized by air. The rate at which it is
released by hydra varies with both temperature
and nutrition, being especially high during
periods of active digestion. Its rate of accumula-
tion varies with both the degree of crowding
and stagnation of the cultures. Active solutions
equilibrated with air transmit their activity to the
gas phase. Although the volatile chemical, or gas,
is as yet finidentified, present methods now permit
the reversible control of sexual differentiation in
hydra for the first time. A similar volatile factor,
capable of inducing sexual differentiation, has

been described in both the Cecropia moth (M. M.

KETCHELL AND CARROLL WiLuraMs, Biol. Bull.

109: 64, 1955) and the crustacea Daphnia (A. M.

Banta, paper No. 39, Dep’t. of Genetics, Carnegie

Inst. of Washington (1939)).

991. Fluorimetry of oxidized and reduced
pyridine nucleotides and microanalytical
applications. OtiverR H. Lowry, Nira R.
Roperts,* Joyce I. KApPHAHN* AND CHARLES
Lewis.* Dept. of Pharmacology, Washington
Univ. School of Medicine, St. Louis, Mo.

With the method of Kaplan et al. (J. Biol.

Chem. 191: 461, 1951) and a suitable fluorimeter

Volume 16

DPN* or TPN*t may be accurately measured at
concentration of 10 m (5 X 107" m in 0.05 ml),
The procedure, modified from Kaplan et al. is to 1)
acidify (to 0.2 Nn HCl) to destroy DPNH or TPNH;
2) let stand 1 hr. at 25 or 30° in 6 N NaOH to
develop fluorescence; and 3) dilute 10-fold with
water to reduce sensitivity to light before reading.
DPNH and TPNH are measured at px 8 to 12 by
native fluorescence, without inducing fluorescence
in DPN* or TPN*. Although the DPNH (TPNH)
method is about 10 times less sensitive than that
for DPNt, it exceeds ultraviolet absorbtion
(340 my) in useful sensitivity by a factor of 200,
Mg can quadruple TPNH fluorescence and in-
crease DPNH fluorescence 50%. By supplementa-
tion with suitable purified enzymes almost any
substrate or enzyme may be directly measured
in minute quantity by formation of either DPN?,
TPN*, DPNH or TPNH. Thus aspartic acid can
be measured in 5 y of brain with a mixture of
alpha-ketoglutarate, glutamic aspartic trans-
aminase, malic dehydrogenase and DPNH, by
measuring the DPN* formed. The sensitivity for
enzyme measurements is a little surprising. For
example, a malic dehydrogenase method requires
only 10-5 y of dry brain (1 or 2 mitochondria) or
approximately a million molecules of enzyme.
(Supported by the American Cancer Society
through the Committee on Growth.)

992. Effects of nucleosides on the resistance of
normal human erythrocytes to osmotic
lysis. Bertram A. Lowy, Ernst R. Jarrt,
Grace A. VANDERHOFF, PHILIP AISEN* AND
Irvine M. Lonpon (introduced by GrorRGE
BoswortH Brown). Depts. of Medicine and
Biochemistry, Albert Einstein College of Medi-
cine, Yeshiva Univ., New York City.

The metabolism of several nucleosides and
related compounds in the normal human erythro-
cyte and the effects of these compounds on its
resistance to osmotic lysis were studied. Freshly
drawn, washed erythrocytes were incubated at
37° for 2 hr. as a 16.7% suspension in isotonic
saline or in pH 7.4 isotonic phosphate buffer con-
taining the compound under study. The cells were
centrifuged, washed and resuspended in saline or
phosphate. Aliquots were subjected to osmotic
stress by addition to hypotonic solutions of saline
or phosphate. The extent of lysis was measured
by the amount of hemoglobin released. Portions
of the supernatant solution obtained after incu-
bation were assayed for ribose or deoxyribose.
Of the compounds studied thus far, adenosine
(2-20 um/ml), inosine (2-20 um/ml), guanosine
(2.4 ywM/ml), 2,6-diaminopurine riboside (10
uM/ml) and deoxyadenosine (10 um/ml) exhibited
a marked protective effect against osmotic lysis,
related in some cases to the concentration of

a nin in wi... on. ee nT

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NH)
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PN*,
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osine

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March 1956

phosphate‘or sodium chloride. During incubation
with these compounds, a considerable uptake of
the sugar moiety occurred. Ribose (4-10 um/ml)
and ribose-5-phosphate (2 uM/ml), alone, or in the
presence of adenine or hypoxanthine had no effect
upon susceptibility to osmotic lysis despite some
uptake of the sugars. On incubation in phosphate
buffer neither protective effect nor significant
ribose uptake was observed with adenosine-2-
phosphate (10 mwM/ml), adenosine-3-phosphate
(10 um/ml), adenosine 5-phosphate (10 um/ml) or
adenosine triphosphate (10 um/ml). Various other
nucleosides, sugars and related compounds tested
showed no protective effect under comparable
conditions.

993. A new intermediate in purine biosynthe-
sis. Lewis N. LuKENsS* AND JOHN M. BucHANAN.
Div. of Biochemistry, Massachusetts Inst. of
Technology, Cambridge.

A study of the conversion of 5-aminoimidazole
ribotide (AIR) to 5-amino-4-imidazole carbox-
amide ribotide (AICAR), one of the steps in the
de novo synthesis of inosinic acid by avian liver,
has shown that the overall reaction has a require-
ment for ATP, aspartic acid and bicarbonate.
Glutamic acid could not replace aspartic acid.
Fractionation of extracts of pigeon liver has now
succeeded in separating the enzymes responsible
for this conversion into 2 components, Fractions
I and II. Neither fraction individually can con-
vert AIR to AICAR. However, when Fraction I is
incubated with all the substrates required for the
overall reaction, an intermediate accumulates.
Upon incubation with Fraction II the inter-
mediate is converted to AICAR plus fumaric or
malic acid (fumarase is present in Fraction II).
No substrates other than the intermediate are
required for its conversion to AICAR. When the
intermediate is synthesized enzymatically in the
presence of 4-C'4 aspartic acid, 1 m of aspartic acid
is incorporated into each mole of the compound.
Aspartic acid is a product of acid hydrolysis of
the new intermediate. The intermediate was
isolated and purified by chromatography on
Dowex-1 resin columns. The compound absorbs
maximally at 266 mu at pH 7. Analytical results
have shown the presence of pentose:organic
phosphate: glycine:acid-labile nitrogen:total or-
ganic nitrogen in the approximate molecular
ratios of 1:1:1:2:4. While the proof of structure is
still incomplete, present data indicate that the
intermediate is 5-amino-4-imidazole-(N-succinylo-
carboxamide) ribotide.

994. Progesterone side-chain oxidation. W. 8.
Lynn, JR. (introduced by Henry Kamtn.)
Depts. of Medicine and Biochemistry, Duke Univ.
School of Medicine, Durham, N.C.

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS 305

An ammonium sulfate fraction from rat and
bovine testicular extracts can cleave the side-
chain of progesterone to acetic acid. One ug of
substrate per half gram of tissue is cleaved per
hour by normal extracts, but extracts prepared
from rats previously injected with chorionic
gonadotropin accelerate this cleavage over a
thousand fold. The enzyme fraction is prepared
by homogenization in isotonic sodium chloride-
bicarbonate buffer (pH 7.4), centrifugation at
100,000 X G for 20 min., precipitation in 40%
ammonium sulfate solution, followed by dialysis
against the bicarbonate buffer for 12 hr. Freezing
destroys the activity. TPN, TPNH, ATP or
fructose 1,6 diphosphate, and O2 are necessary for
maximal activity. Using 21-C'* progesterone, 2
probable intermediates have been isolated; 17-«
hydroxy progesterone, and a A4,3 keto pregnene
17,20 glycol. This glycol has an Rf slightly greater
than Compound B in the Zaffaroni paper chroma-
tographic system and was identified as 17,20
glycol by obtaining acetaldehyde and a 17 keto
steroid after periodic oxidation (Appleby and
Norymberski). Its ultraviolet absorption indi-
cates that it is a A4 ketone. The nucleotide re-
quirements appear to be necessary for the steps
leading to the glycol formation, whereas Oz is
necessary for the cleavage reaction. Specifically,
TPNH is necessary for the reduction of the 17a
hydroxy progesterone to the 17,20 glycol. Al-
though the hydroxylating enzymes have not yet
been separated from the cleaving enzymes, some
of the data suggests that acetic acid may not be
the actual cleavage product. By using the 2 inter-
mediates as trapping agents, it can be shown that
the sequence of events is: progesterone — 17a OH
progesterone — 17,20 glycol — acetate.

995. Mechanism of the enzymatic synthesis of
pantothenate from beta-alanine and pan-
toate. W. K. Maas. Dept. of Pharmacology,
New York Univ. College of Medicine, New York
City.

The synthesis of pantothenate, catalyzed by an
enzyme system from Escherichia coli, involves the
following over-all reaction: pantoate + beta-
alanine + ATP — pantothenate + AMP + in-
organic pyrophosphate (PPi) (Maas, Federation
Proc. 12: 241, 1953). The results outlined below
indicate that the formation of this peptidic
linkage proceeds via enzyme-bound pantoyl ~
AMP, according to the scheme: 1) ATP + pantoate
+E = E-pantoyl ~ AMP + PP;; 2) E-pantoyl ~
AMP + beta-alanine — pantothenate + AMP +
E. 1) The 50-fold purified enzyme catalyzes a
pantoate-dependent incorporation of P**-PP; into
ATP. 2) C!4-AMP is not incorporated into ATP
in the presence of any of the substrates or products
of the reaction. 3) Formation of a pantoyl com-

306 FEDERATION PROCEEDINGS

pound as intermediate is shown by the production
of equimolar amounts of pantoylhydroxamic acid
and PP; on incubation of enzyme with ATP,
pantoate and hydroxylamine. 4) Binding of this
compound to the enzyme is suggested by the
observation that incubation of enzyme, ATP and
pantoate, even with pyrophosphatase to pull the
reaction, fails to yield pantoylhydroxamic acid,
as tested by subsequent addition of hydroxyl-
amine. 5) More direct evidence for anhydride
formation between pantoate and AMP is pro-
vided by the finding, obtained in collaboration
with Yarmolinsky and Koshland, that during
pantothenate synthesis 1 atom of O" is trans-
ferred from the carboxy] group of pantoate to the
phosphate of AMP.

996. Metabolism of taurine in the growing
chicken. L. J. Macuiin (introduced by N. R.
Exits). Animal and Poultry Husbandry Research
Branch, Dept. of Agriculture, Beltsville, Md.
Female chickens were injected at 3 wk. of age

with 94 ue of taurine-S**. After 1 day 16% of the
S*5 administered was recovered in the excreta.
Over a period of 9 days following the injection
about 39% was recovered in the excreta. At the
end of this period 55% was found in the carcasses.
The radioactivity in various tissues in counts/
min/gm fresh tissue X 10-* at 1 day was as fol-
lows; heart, 1295; duodenum, 859; spleen, 771;
gizzard muscle, 648; proventriculus, 483; liver,
440; leg muscle, 340; pancreas, 290; kidney, 169;
blood, 120; brain, 65; breast muscle, 35. About
90% of the total S** present in the heart and
gizzard was accounted for as taurine-S* and
about 6% and 3%, respectively, as taurocon-
jugated bile acids. In the liver and kidney how-
ever, less than 50% of the total S** was present
as taurine-S** and 4% and 1%, respectively, as
bile acids. The remainder of the S** in the tissues is
present gs compounds other than sulfate, cysteic
acid or cysteinesulfinic acid. These radioactive
compounds have not yet been identified. The
results suggest that in the chicken, taurine is not
an inert product of the oxidation of the sulfur-
containing amino acids, but may have metabolic
functions in addition to serving as a precursor of
bile acids.

997. Effect of cortisone, stress and sulfhydryl!
precursors on thiourea pleural effusion.
Jutta B. MACKENZIE AND Cosmo G. MACKENZIE.
Dept. of Biochemistry, Univ. of Colorado School
of Medicine, Denver.

Adult rats develop massive pleural effusion
when given a dose of 1 mg/kg of thiourea. Im-
mature rats are completely resistant to more than
three thousand times this dose. Since the onset of
susceptibility to thiourea coincides with puberty,

Volume 1§

the role of sex hormones and other endocrine
secretions in pulmonary edema was investigated,
Pleural effusion was produced in many of the
thiourea-treated young by the administration of
either ACTH, cortisone, hydrocortisone and
fluorohydrocortisone. This effect of cortisone was
observed in 50% of the young and (although all of
the adults are highly susceptible to pleural effu-
sion) was litter specific. Pleural effusion was also
produced in susceptible litters by exposing the
thiourea treated young to the stresses of heat,
cold or altitude. The administration to immature
rats of desoxycorticosterone acetate, testosterone,
thyroxin, growth hormone, etc., together with
thiourea was without effect. When adult rats were
given cortisone the lethal dose of thiourea was
reduced by approximately one-half. Nevertheless,
removal of the adrenals, either alone or in con-
junction with castration, gave no protection
against thiourea toxicity. Immunity to thiourea
toxicity was produced by giving adult rats minute
doses of thiourea over a 3-day period prior to the
otherwise lethal dose. In addition, cysteine
and homocysteine detoxify thiourea in the mature
animal. Even more potent as a detoxifying agent
was the sulphur containing ring compound,
thiazolidine carboxylic acid, which is a source
of metabolically generated SH groups in mito-
chondrial preparations (Federation Proc. 14:
223, 1955) and in the intact animal. Thiazolidine
was likewise highly effective.

998. Sodium and potassium requirements of
marine bacteria. Ropert A. MacLeop anp
Eve Onorrey.* Pacific Fisheries Expl. Station,
Vancouver, Canada.

The Na* requirement of 3 marine bacteria for
maximum rate of growth was found to be .1-—.15
mM/ml of medium or about one-quarter of the
concentration of Nat in sea water. Maximum
growth could also be achieved with half this
amount of Na* if the incubation period was
sufficiently long. Below a level of about .05 mm/ml
the growth response of the organisms to Na+ was
proportional to the amount of Nat added ir-
respective of the length of the incubation period.
Neither Lit, K*, Rb*+ nor Cs* was able to replace
the requirement for Na*. Although all except Cs*
stimulated early growth of the organisms at sub-
optimal levels of Nat, the sparing action of
these ions on the Na* requirement was not ap-
parent after longer incubation periods. When
growth was limited by the Nat level in the medium,
analysis of the medium after growth had ceased
revealed that no appreciable change had occurred
in the Na* concentration. Although the amount
of Nat removed by the cells relative to the total
present in the medium was small, analysis of cell
preparations revealed an actual concentration of

tre
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rine
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e was
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ature
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ourea
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ot ap-
When
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curred
mount
> total
of cell
ion of

March 1956

Nat by the cells. The organisms require about
one-hundredth as much K* as Nat for growth.
The K+ requirement is similarly specific. When
growth was limited by the K* level, only a frac-
tion of the available potassium in the medium
had been taken up when growth had ceased,
though again K* was found capable of being
concentrated by the cells.

999. Effect of deuterium substitution on the
kinetics of hydrogen transfers catalyzed by
pyridinoproteins. H. R. MAHLER AND JOYCE
Dovetas.* Dept. of Chemistry, Indiana Univ.,
Bloomington.

In an effort to elucidate the mechanism of
enzyme-mediated hydrogen transfer reactions,
the influence of substitution of deuterium for
hydrogen in DPNH on the kinetics of the reaction
catalyzed by different pyridinoproteins have been
studied. Aleohol dehydrogenase (yeast) (I), and
lactic dehydrogenase (heart muscle) (II) were
chosen for the initial investigation since kinetic
evidence previously adduced (Hayes and Velick,
Schwert and Hakala), indicates binding of sub-
strate and coenzyme at 2 separate, distinct and
independent sites. Variation of reactant concen-
trations (DPNH and DPND, oxidized sub-
strates; in 0.1 m phosphate, pu 7.0 and 7.9 for I,
pH 7.0 for II) affected the kinetics as follows: a)
the Michaelis constants for reduced coenzyme,
determined at different substrate concentrations
varying by a factor of 10, were constant, but
Kppna = 1.75 Kppnp; 6) K*>stt showed a similar
constancy over a corresponding range of co-
enzyme concentration but was independent of the
substitution of deuterium into the pyridine
nucleotide; c) Vmax (DPNH— ~, substrate > ~)
determined by several methods also showed the
ratio of 1.75:1 for the reactions of DPNH com-
pared to DPND. These results indicate 1) that,
since at least in the case of I Michaelis and dis-
sociation constants for I-DPNH are essentially
equal, there is no effect of the hydrogen para to the
N (Colowick and San Pietro) on the binding of
reduced coenzyme by the enzyme; 2) binding of
substrate is indeed independent of the concentra-
tion of coenzyme; 3) the direct, stereo-specific
transfer of the para hydrogen known to occur in
these reactions (Vennesland, Westheimer ei al.)
probably takes place in the rate-limiting step
and within a ternary complex of enzyme coenzyme
and substrate.

1000. Isolation of chenodeoxycholic acid
metabolite from the urine of surgically
jaundiced rats. THEODORE A. MaHowaALp*
AND Wiiu1AM H. Etiort. Dept. of Biochemistry,
St. Louis Univ. School of Medicine, St. Louis, Mo.
Of the 2 metabolites reported after administra-

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS 307

tion of radioactive chenodeoxycholie acid, the less
polar substance was found to be identical with an
acid (Acid I) obtained in very small quantitn
from normal rat bile (Mahowald et al. Federatioy
Proc. 14: 250, 1955). After administration of
radioactive chenodeoxycholic acid to bile duct
ligated rats, the radioactivity was excreted in the
urine. Chromatography of the hydrolyzed urine
yielded in the main radioactive zone a crystalline
acid identical with the less polar acid (Acid I)
found in bile. From the radioactivity data, it can
be calculated that 35 mg (average of 3 rats) of this
acid are excreted in the urine during the first 72 hr.
after duct ligation and administration of radio-
active chenodeoxycholic acid whereas only 20 mg
of cholic acid are excreted during the same period.
During the first 72 hr. following duct cannulation,
80 mg of cholic acid and 2-3 mg of Acid I are ex-
creted in the bile. These observations show a
quantitative difference in the metabolism of
chenodeoxycholic acid as well as providing a
means for obtaining large quantities of the rare
Acid I.

1001. Normal and thyrotoxic rat liver mito-
chondria. Guapys FreLpotr MAtey (intro-
duced by W. H. Peterson). Inst. for Enzyme
Research, Univ. of Wisconsin, Madison.

In order to determine the mechanism by which
the thyroid hormone regulates tissue metabolism
a number of reactions have been studied in normal
and thyrotoxiec rat liver mitochondria. Significant
increases in the levels of succinoxidase, cyto-
chrome c, cytochrome oxidase, and in the oxida-
tion of Krebs cycle intermediates occur in thyro-
toxicity. No change in ATPase as measured in
fresh mitochondria was found although this
enzyme is activated by shorter ‘preaging’ periods
in thyrotoxic mitochondria than are necessary for
normal. In addition activation by Cat*+ and Mgt+
or by DNP yields an apparent increase in ATPase
potential in thyrotoxic mitochondria. In vitro
studies on the oxidation of certain substrates by
thyrotoxic rat liver mitochondria showed a
definite requirement for added nucleotides. The
acid soluble nucleotides of mitochondria from
normal and thyrotoxic rats livers were separated
by chromatography and measured by their Eng
absorption. The release of nucleotides from the
mitochondria of these metabolically different
animals has also been studied and the results may
indicate a difference in permeability as does the
release of ATPase.

1002. Incorporation of 8-azaguanine-4-C'
into bacterial nucleic acids. H. Grorar
ManveEt. Dept. of Pharmacology, The George
Washington Univ. School of Medicine, Washing-
ton, D. C.

308

The incorporation of the radioactive purine
analog, 8-azaguanine-4-C'*, into a microorganism,
Bacillus cereus, was studied in a medium of casein
hydrolysate and salts. The analog was incorpo-
rated only when added to actively dividing cells
and produced significant inhibition of growth.
No effect on gross morphology or viability of the
organisms was observed. Various purine com-
pounds, when added to the bacterial media con-
taining azaguanine, decreased the incorporation of
the analog but did not displace it altogether and
simultaneously reversed the inhibition. When
grown in azaguanine-supplemented glucose-salts
media the microorganisms were only slightly
inhibited and incorporated little azaguanine. The
addition of aspartate to the unsupplemented
glucose-salts media increased the growth rate and
after the azaguanine supplement the relative
inhibition was greater. The incorporation of the
analog also was increased by the addition of
aspartate to the medium. Fractionation of the
bacteria showed that the major portion of the
radioactivity was in the nucleic acids, and a small
amount in the TCA-soluble fraction. After dis-
integration of the bacterial cells by vibration,
followed by salt extraction and degradation to the
nucleotides, radioactivity was recovered almost
quantitatively in a single fraction by elution with
5 m formic acid on Dowex-1, after the emergence
of the normal nucleotides. At 20 mg of azaguanine
per liter of casein hydrolysate medium approxi-
mately 3% of the azaguanine supplied was in-
corporated within 3 hr., mainly into the RNA.

1003. Structural studies on lactobacillic acid
and other long-chain fatty acids contain-
ing the cyclopropane ring. Gino J. Marco*
AND Kuiaus Hormann. Biochemistry Dept.,
Univ. of Pittsburgh School of Medicine, Pitts-
burgh, Pa.

Lactobacillic acid, a major constituent of the
lipides of*several lactobacilli, was shown to con-
tain a cyclopropane ring attached to a straight
chain of 18 carbon atoms (HOFMANN, JUCKER,
MILLER, YounG AND Tausie, J. Am. Chem. Soc.
76: 1799, 1954). The exact location of this ring
remained to be determined. A degradation method
has now been developed locating the cyclopropane
ring in the 11,12-position. This result in conjunc-
tion with our previous findings establishes lacto-
bacillic acid as 11,12-methyleneoctadecanoic acid.
The spatial configuration of the compound re-
mains to be worked out. The degradation method
involves 1) rupture of the 3-membered ring by ex-
posure of the acid to the action of hydrogen bro-
mide in glacial acetic acid; 2) dehydrobromina-
tion of the ensuing bromo acids with collidine;
$) oxidative cleavage of the resulting unsaturated,
branched-chain acids; and finally 4) chromato-

FEDERATION PROCEEDINGS

Volume 165

graphic separation of the split fragments (mono-
and dicarboxylic acids) on suitably buffered silicic
acid columns. The chromatographically homo-
geneous dicarboxylic acid fragments were identi-
fied by melting and mixed melting point deter-
minations and by matching their x-ray diffraction
patterns with those of authentic samples. The
nature of the dicarboxylic acid fragments fixes
the location of the cyclopropane ring (acids with
the ring in the 9,10-position afforded azelaic acid;
those possessing the ring in the 11,12-position,
undecanedioic acid).

1004. Formation of lactic and fumaric acid by
Rhizopus MX. Maurice Mareutiss (intro-
duced by Wo.ur Visunrac). Dept. of Micro-
biology, Yale Univ., New Haven, Conn.

The phycomycete Rhizopus, strain MX, has
been reported by Waksman and Foster (J. Agr.
Research, 57: 873, 1938) to produce more lactic acid
in the dissimilation of glucose under aerobic than
under anaerobic conditions. This stimulation of
lactic acid formation by oxygen indicated the
possibility of a novel mechanism of lactic acid
synthesis. Attempts to reproduce the results of
the above-mentioned authors confirmed that
carbon dioxide, ethanol and lactic acid were
formed anaerobically from glucose. However,
aerobically no increase in lactic acid was noted,
although total activity increased markedly. The
additional acid formed in air could largely be
accounted for as fumaric acid, small amounts of
other acids were also formed. It is suggested that
the original report of oxygen stimulated lactic
acid production was based on insufficiently specific
analytical methods and that no new mechanism of
lactic acid formation need be postulated. The
study of Gibbs and Gastel (Arch. Biochem. &
Biophys. 43: 33, 1953) on the dissimilation of
labeled glucose by a different strain of Rhizopus
indicated glycolysis as a major metabolic pathway.
Their conclusions are supported by our demon-
stration of a number of glycolytic enzymes,
including a DPN-linked lactic dehydrogenase, in
extracts of Rhizopus MX. Aerobically appreciable
amounts of C!4Oz are fixed.

1005. Chromatographic studies of tissue
phospholipides. G. V. Marinerti,* R. F.
Witter AnD E. Srotz. Dept. of Biochemistry,
Univ. of Rochester School of Medicine and Den-
tistry, Rochester, N. Y.

Chromatographic solvents which were devised
for the separation of model phospholipide mix-
tures (WiTreR, MARINETTI AND Storz, Federa-
tion Proc. 1956) were found to be excellent for the
resolution of more complex phospholipide mix-
tures obtained from animal tissues. An investiga-
tion was made of the in vivo incorporation of P®

me 1§

nono-
silicic
1omo-
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The
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ie
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March 1956

into the individual phospholipides of liver,
heart, intestine, kidney, lung, spleen and brain
of the rat. Radioautograms of these tissue lipides
showed intense spots for lecithin and cephalin and
weaker spots for lipide components corresponding
to sphingomyelin, acetal phosphatide, serine
phosphatide, inositol phosphatide and lysolecithin.
Phosphatidic acid was not found in any of the
tissues studied. Other radioactive components
did not behave like any of the known phospho-
lipides studied and are believed to represent new
types of phospholipides. Significant differences
were observed in the composition of the labeled
phospholipides from these tissues. The diisobutyl
ketone-acetic acid-water solvent, employing
silicic acid impregnated paper, resolved the tissue
phospholipides into the largest number of com-
ponents. It was also found that the addition of
water to the chloroform-methanol solvent re-
ported by Lea, Rhodes and Stoll (Biochem. J.
60: 353, 1955) significantly improved this system.
For 2 dimensional chromatography, the use of
chloroform-methanol-water followed by diisobutyl
ketone-acetic acid-water proved excellent. The
chromatographic methods were also successfully
used for the study of products obtained by frac-
tionation of tissue lipides on silicic acid columns
and by the action of lecithinases on lecithin.

1006. Effect of diet and adrenalectomy on
proteinuria in experimental nephrosis.
Jutran B. Marsu anp Davin L. DraBkIn.
Dept. of Biochemistry, Grad. School of Medicine,
Univ. of Pennsylvania, Philadelphia.

The proteinuria of nonedematous rats (50-100
gm in weight) was found to depend directly on the
protein content of the diet. The average protein
excretion on synthetic diets containing 5, 22 and
33% of protein was, respectively 8, 115 and 174
mg/day. Rats on the lowest protein diet, when
shifted to the 33% protein diet responded with an
immediate increase in proteinuria, reaching the
maximum within 48 hr. Substitution of fat for
carbohydrate on the highest protein diet dimin-
ished proteinuria but the consumption of food
was similarly decreased. When rats with estab-
lished nephrosis were adrenalectomized and
maintained on 0.9% saline in the drinking water,
they excreted 15% less protein on a standard 25%
protein diet, but the daily food consumption was
also 15% lower. However, when adrenalectomized
and control nephrotic rats were fasted overnight,
the drop in protein excretion during the fasting
period was considerably greater in the adrenalec-
tomized animals. These findings suggest that the
synthesis of plasma albumin from endogenous
amino acids is more limited after adrenalectomy
than in the intact nephrotic animal, possibly be-
cause of a defect in amino acid mobilization. The

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

309

livers of severely nephrotic rats were found to
have hypertrophied, and this was accompanied
by a 30% increase in liver RNA.

1007. An intermediate from succinate 2,3-C!4.
Lawrence M. MarsHALL AND CLARENCE
VauGun.* Dept. of Biochemistry, Howard Univ.
College of Medicine, Washington, D.C.

When succinate 2,3-C-!4 was incubated with
whole homogenates of liver from 11-, 14- and 17-
day old chick embryos, a compound appeared
which was not produced when carboxyl-labeled
succinate was the substrate. The chromatographic
mobility, on paper and on silica gel columns, did
not allow the possibility that the elaborated
product was a known substrate of the tricarboxylic
acid cycle. The intermediate appeared when the
cycle was blocked with malonate. Neither fumaric
acid 1,4-C'4 nor citric acid 1,5-C!4, when incu-
bated with the homogenates for 20 min., served as
precursors for the intermediate. The isolated
intermediate did not react with 2,4 dinitrophenyl-
hydrazine. Difference between the distribution
coefficients in ether and hydrochloric acid (0.1 Nn)
and in ether and aqueous sodium hydroxide
(0.1 n) did not characterize the compound as an
acid. Relative densities of the spots designating
the dicarboxylic acids and the intermediate
on radioautographs from extracts of homogenates
incubated with the several labeled organic acid
substrates indicate that the methylene carbons
exclusively are the precursors of this decarboxyla-
tion product of succinic acid.

1008. Trans-L-epoxysuccinate as an inhibitor
of bacteria, and its reversal. Witiiam R.
Martin* aND J. W. Foster. Dept. of Bac-
teriology, Univ. of Texas, Austin.

Endogenous respiration of washed cells of
bacteria was completely inhibited by low con-
centrations of Na trans-L-epoxysuccinate (TES).
Molar equivalents of citric acid cycle inter-
mediates were rapidly oxidized. Using the spread
plate procedure with glucose-salts agar (FosTER
AND Pitti.Luo, J. Bact. 65: 361, 1953), as little as
50 wg of TES per ml of medium completely in-
hibited growth of several representative bacterial
species in a 24 hr. incubation period. Longer
incubation required higher concentrations. Several
organic acid analogues of TES were tested for their
ability to reverse the toxicity of TES for Escher-
ichia coli. Succinate (20 ug) completely reversed
the inhibition caused by 400 ug TES. Fumarate
had slight reversal properties and malate none.
A mixture of all the common B vitamins failed to
reverse, as did a nontoxic mixture of purines and
pyrimidines. Complex organic substances (100
ug), such as yeast extract, corn steep liquor, etc.,
completely reversed the toxicity, indicating they

310

contain a reversing substance(s) considerably
more potent than succinate. TES (100-250 um)
inhibited the oxidation of 10 um of succinate by
beef heart succinic dehydrogenase; the inhibition
was only partially reversed by excess succinate.
Under comparable conditions malonate was about
10 times more inhibitory.

1009. Mechanisms of enzyme-catalyzed oxygen
transfer. H. S. Mason anp I. ONOPRIENKO.
Dept. of Biochemistry, Univ. of Oregon Med.
School, Portland.

In order to throw some light upon mechanisms
of action of oxygen-dependent metallo-protein
oxidases, we have determined the source of the
oxygen which appears as hydroxyl in substrates
of the model hydroxylase, ferrous-EDTA-O:-
ascorbate, using O" as a tracer. We find that hy-
droxyl oxygen so introduced (into salicylate)
arises from molecular oxygen. Since these ascor-
bate-coupled Fenton hydroxylations occur ex-
clusively at electronegative sites upon aromatic
rings (unlike hydroxylations due to free radicals),
the hydroxylating species appears to be the
chelate of a cation containing oxygen derived
from the atmosphere, i.e. ferryl ion, Fe(OQH)***+ =
FeO** + H*, formation of which from molecular
oxygen requires an electron source. Oxygen-
dependent monohydroxylating oxidases require,
similarly, specific electron sources (e.g. TPNH,
DPNH, DOPA, ascorbate) which appear to serve
a related function.

1010. Further characterization of the kynure-
nine transaminase of rat kidney. MERLE
Mason (introduced by Lina MILER). Dept. of
Biological Chemistry, Univ. of Michigan, Ann
Arbor.

Rat kidney which is homogenized in slightly
acidic phosphate buffer (px 6-7) loses kynurenine
transaminase activity rapidly unless a-ketoglu-
tarate #& present in the homogenate at a concen-
tration of 4 um/ml or greater (Mason, J. Biol.
Chem. 211: 839, 1954). This activity can be re-
stored completely by the addition of catalytic
amounts (10-20 um/ml) of pyridoxal-5-phosphate.
Homogenates thus reactivated become inactive
again on dialysis overnight in 20 volumes of 0.1 M
phosphate buffer, pn 6.3. If a-ketoglutarate is
present in the homogenate and buffer during
dialysis, only a relatively small degree of inactiva-
tion occurs. These observations are interpreted to
indicate that the kynurenine transaminase of rat
kidney is readily dissociable in slightly acidic
solutions into apoenzyme and a coenzyme which
is identical with or closely related to pyridoxal-5-
phosphate and that a-ketoglutarate in some way
prevents the dissociation. The possibility is con-
sidered that a-ketoglutarate prevents inactiva-

FEDERATION PROCEEDINGS

Volume i

tion by combining with the holoenzyme by means

.of Schiff’s base formation with a pyridoxamine

phosphate moiety, the Schiff’s base having 4
greater affinity for the apoenzyme. Pyruvate
does not prevent inactivation at concentrations
of 4 um/ml and does not transaminate with kynu-
renine under conditions which are optimal for the
kynurenine-a-ketoglutarate transamination.

1011. Fumaric hydrogenase activity of suc.
cinic dehydrogenase. VINCENT Massey,’
THomas P. SrinceR AND Epna B. Kearney,
Edsel B. Ford Inst. for Med. Research, Henry
Ford Hosp., Detroit, Mich.

Since the reoxidation of hydrosulfite-reduced
beef heart succinic dehydrogenase is accom-
plished very rapidly by the addition of fumarate,
it would be expected that succinic dehydrogenase
could function catalytically as an efficient fumaric
hydrogenase. For measurement of the activity,
the general method of Fischer et al. (Ann. Chem.
552: 203, 1942) has been used. With the system,

, P succinic
fumarate + leucodiethylsafranin ———————>
dehydrogenase

succinate + diethylsafranin, in which leuco-
diethylsafranin serves to reduce the oxidized
enzyme, and the color of diethylsafranin is meas-
ured, the properties of the ‘fumaric hydrogenase’
reaction have been studied with highly purified
preparations of succinic dehydrogenase. Like the
forward reaction, the conversion of fumarate to
succinate is greatly accelerated by the addition
of phosphate, strongly inhibited by p-chloro-
mercuribenzoate and malonate, and the pH
optimum is very similar (pH 7.5). Under the con-
ditions used the maximum velocity of the reverse
reaction is about 4% oth the velocity of the forward
reaction.

1012. Sedium chondroitin sulfate-protein
complex from cartilage. MarTIN B. MaTHEWws
AND IRENE LozartyTEe.* Depts. of Pediatrics
and Biochemistry, Univ. of Chicago and Ia
Rabida Jackson Park Sanitarium, Chicago, Ill.
Sodium chondroitin sulfate-protein complex

(SCS-P) was extracted from bovine cartilage at

neutral pH and purified in various ways. The

composition of the ‘anhydrous’ preparation is

variable with a SCS content from 60 to 75%.

Hydroxyproline varies from less than 0.05-0.24%

and is probably due to trace contamination with

collagen. Tyrosine values are from 0.9 to 1.4%

and protein contents are estimated from 25 to

40% for the different preparations. These tyrosine

values are much lower than those reported for

similar preparations (‘mucoprotein’ of SHATTON

AND ScuuBeEertT, J. Biol. Chem. 211: 565, 1954).

Inhomogeneity of average composition is also

shown by separation of fractions of different SCS

dit

lume 1§

’ Means
xamine
ving 4
Tuvate
rations
L kynu-
for the
-

f suc.
\SSEY,*
ARNEY,

Henry

educed
Accom-
narate,
genase
umaric
tivity,
Chem.
ystem,
nic

——+
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meas-
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urified
ke the
‘ate to
ldition
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e con-
everse
yrward

rotein
THEWS
iatrics
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. The
ion is
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0.24%
n with
. 1.4%
25 to
Tosine
ed for
ATTON
1954),
s also
it SCS

March 1956

to protein ratio in preparative ultracentrifuge
experiments. Molecular weights (Mw) from
angular light scattering data are from 2 X 10° to
8 X 10°. The scattering envelopes are consistent
with polydisperse systems of rods with a linear
density about 1000 avogram per A. (Aided by the
Chicago Heart Assoc.)

1013. Phosphorylation efficiency in rabbits
fed a tocopherol -deficient diet. Paut McCay*
AND RANWEL Caputto. Oklahoma Med. Research
Fndn, Oklahoma City.

Three groups of rabbits were studied: (J) on a
balanced ration, (IZ) on the tocopherol-deficient
diet of Mason and Harris, (JZJ) on this diet plus
a-tocopheryl acetate. The deficient diet produced
sterility but allowed normal growth in rats. As
was expected, (JJ) did not develop muscular
dystrophy nor the high creatine/creatinine ratios
seen in (JJ) but neither of these 2 groups grew
normally. Furthermore, (J/J) developed a curious
fur-eating habit not seen in (7J). At killing, after
55-100 days on the ration, paradoxically (III)
had more severely fatty, fibrotic livers than (JJ).
P/O ratios obtained from oxidation of a-keto-
glutarate by liver mitochondria gave the following
means: (J), 2.70; (Z7), 1.60; (II), 2.28. For the
difference between (7) and (JJ), P < .01, and
between (IJ) and (III), P < .02. To localize the
biochemical lesion in the coupling of phosphoryla-
tion which occurs in (JJ), experiments were done
adding 1 of the following components to the reac-
tion mixture: dinitrophenol; K;Fe(CN). (anaero-
biosis); or Antimycin A, ascorbate and excess
cytochrome c. Results failed to show differences
between the 3 groups in these experiments. It is
concluded that the diet has a deficiency besides
vitamin E which causes liver damage, and that the
addition of vitamin E aggravates this effect of the
deficiency. Lack of vitamin E results in a condi-
tion of the liver such that the efficiency of oxida-
tive phosphorylation is diminished, but the
failure to localize the site of the uncoupling leaves
it inconclusive whether it is a specific effect or due
to a nonspecific damage of the liver mitochondria.

1014. Fractionation of fluorine in milk. J. F.
McCLENDON AND J. GERSHON-COHEN.* Albert
Einstein Med. Ctr., Northern Div., Philadelphia,
Pa.

Since it was shown by the senior author (Federa-
tion Proc. 3: 94, 1949) that rats on a fluorine-free
diet lost their molar teeth due to extensive caries
and died and that milk would prolong their life,
the question has been asked whether the fluorine
or some other constituent in milk prolonged their
life. Hence milk was separated in a Sharpless
super-centrifuge at 50,000 rpm for 1 hr. and the
butter fat that rose to the top, the whey that

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

311

remained in the middle and the casein that pre-
cipitated and stuck to the bowl were analyzed for
fluorine by William C. Foster. The results of
several determinations on Philadelphia milk
were: butter fat .76-148 ppm fluorine, whey
.19-.82 (dry basis) and casein .9-1.7 (dry basis).
Hence, it was not possible to add any of these
nutrient fractions to the fluorine-free diet without
adding fluorine.

1015. Time-distribution of Selenium-75 in
dog serum proteins as determined by paper
electrophoresis. KENNETH P. McCoNnNELL,
CuHarRLES H. Wasnitz* anp Dorotuy Roru.*
Radioisotope Service, VA Hosp., Louisville, Ky.
Previous studies (J. Biol. Chem. 212: 747, 1955)

demonstrated that in serum proteins from dogs

injected 24 hr. previously with Se-75 Cl, and
resolved by paper electrophoresis, 60% of the
total strip activity was found equally distributed
between the alpha-2 and beta-1 fractions. About
10% was found in each the albumin, alpha-l,
beta-2 and gamma fractions. Recent experiments
were designed to determine whether quantitative
difference in the selenium protein binding phe-
nomenon existed as related to time. Normal
healthy dogs were injected subcutaneously with
trace amounts of Se™Cl, and samples of blood
were taken at various time intervals up to 48 hr.
after injection. The serum was isolated, stripped
and processed as before. Within 10 min., 30% of
the total strip count was present in the albumin
fraction, rising within an hour to 42%, thereafter
decreasing gradually to about 10% at 48 hr. How-

ever, the alpha-2 and beta-1 fractions were 7%

and 18%, respectively, after 10 min. There was a

gradual increase in the alpha-2 and beta-1 frac-

tions up to 48 hr., where the alpha-2 and beta-1
were found to be 26% and 30%, respectively. The
results were interpreted to indicate that selenium
is first bound chiefly to albumin and later is
transferred or principally bound to _ specific
globulin fractions, namely alpha-2 and beta-l.

The significance of these findings is not known at

present. Perhaps albumin acts as a transport of

selenium as is thought to be the case for copper

and cobalt (Horst, Klin. Wochschr. 32: 961, 1954).

1016. Amino acid requirements of the Walker
256 carcinosarcoma in vitro. THomas A.
McCoy anp Rosert E. Neuman (introduced by
Simon H. WENDER). Biomedical Div., The
Samuel Roberts Noble Fndn., Inc., Ardmore,
Okla.

A routine procedure for culturing Walker 256
carcinosarcoma cells in vitro has been described
(J. Nat. Cancer Inst. In press). The cultures were
established from cell suspensions prepared di-
rectly from the freshly excised tumors. The

312

medium was 5% rat serum, 0.05% yeast extract
and a mixture of amino acids and vitamins in
Earle’s balanced salt solution. Recent studies
have identified L-asparagine as the growth pro-
moting constituent of yeast extract. A require-
ment for both L-glutamine and L-asparagine was
shown. A new basal medium consisting of 5%
dialyzed human serum, an amino acid and vitamin
mixture in Earle’s salt solution was used to deter-
mine the amino acid requirements of the tumor.
Omission of any one of 14 amino acids, including
glutamine and asparagine, resulted in failure to
grow and death of the Walker 256 cell under
tissue culture conditions.

1017. Pyrophosphate exchange and active
luciferin formation by firefly luciferase.
W.D.McEtroy anp Arpa A. GREEN. McCollum-
Pratt Inst., Johns Hopkins Univ., Baltimore, Md.
Crystalline firefly luciferase catalyzes a reac-

tion between luciferin and adenosine triphosphate

which, in the presence of oxygen, eventually leads
to the formation of light, pyrophosphate, adenylic
acid and oxyluciferin. The initial reaction, which
can take place anaerobically, leads to the forma-
tion of pyrophosphate and apparently adenyl-
luciferin. Oxyluciferin will also function in this
pyrophosphate releasing reaction. Consequently,
the addition of pyrophosphatase results in the
continuous breakdown of ATP to inorganic phos-
phate even after light emission has ceased. Ap-
parently the adenyl-oxyluciferin is labile and is
broken down to oxyluciferin and adenylic acid.
When the reaction is allowed to proceed for a
short time in the presence of P32 labeled pyro-
phosphate there is a rapid incorporation of pyro-
phosphate into the residual ATP, indicating
reversibility. Luciferin is required for the in-
corporation of pyrophosphate. These results and
others demonstrate that the initial reaction in
firefly luminescence is as follows: luciferin +
ATP = gdenyl-luciferin + pyrophosphate.

1018. Amino acid incorporation into muscle
protein. JoHN R. McLean,* GrorceE L. Conen*
AND ME vin V. Simpson. Dept. of Biochemistry,
Yale Univ., New Haven, Conn.

In previous in vivo studies, comparable rates
were obtained for the incorporation of labeled
amino acids into the proteins of the microsomal
and mitochondrial rich fractions of rat skeletal
muscle (Biochem. et biophys. acta. In press). These
results are in contrast to those reported for liver,
where the incorporation into mitochondrial pro-
tein is markedly lower than into microsomal
protein. Further in vivo experiments confirming
and extending these results on muscle were per-
formed in which the rate of incorporation of
pt-leucine-1-C!4 or pui-phenylalanine-3-C!* into

FEDERATION PROCEEDINGS

Volume 1§

the proteins of subfractions of these preparationg
was measured at time intervals from 1 to 60 min,
after injection. Treatment of the microsomal
preparation with deoxycholate yielded 2 fractions
separable by centrifugation at 78,000 X g for 3 hr,
The sedimentable fraction incorporated amino
acid at arate 2-3 times that of the nonsedimentable
fraction, a finding similar to the results obtained
in liver by Littlefield et al. (J. Biol. Chem. 217: 111,
1955). The mitochondria-rich preparation, which
also contains an opaque particle peculiar to
muscle, was further separated into 5 fractions by
differential centrifugation at intervals between
2000 X g and 4400 X g. The proteins of all these
fractions showed rapid rates of incorporation of
the labeled compound. The specific activities
of the more active of these fractions were nearly
equal to that of the sedimentable subfraction of
the microsomes. In in vitro experiments, mito-
chondria incorporated labeled leucine at ap-
proximately the same rate as did microsomes.
The results of these experiments suggest a role in
protein synthesis, in muscle, for both microsomes
and mitochondria.

1019. Biological methylation of anserine. I,
RosaBELLE McManvs (introduced by W1LLaRp
A. Kren). Radioisotope Service, VA Hosp.,
West Haven, and Dept. of Biochemistry, Yale
Univ., New Haven, Conn.

Transmethylation of muscle anserine from
methionine was first demonstrated in the rabbit
(J. Biol. Chem. 149: 355, 1943). The present work
is a further study of this reaction in the rat.
Normal young adult rats were injected intra-
peritoneally with 2 ue (10 um) of C!4H;-methionine
and 2 rats were killed at 4, 8, 16, 24 and 48 hr.,
respectively. Deproteinized muscle extracts were
prepared from striated leg muscle and C!* in-
corporation determined. Uptake of C!* in the
extracts was maximal at 24 hr., decreasing slightly
after 48 hr. The deproteinized extracts were
subjected to displacement chromatography on
Dowex 50, H+ columns, using pyridine to displace
neutral and acidic amino acids and creatine
followed by collidine to displace the imidazolyl
constituents. The latter were chromatographed
on Dowex 50, NH,* columns and eluted with 0.2
and 0.5 M ammonium acetate, pH 3.9, accomplish-
ing separation of a_histidine-methylhistidine
fraction, carnosine and anserine. Identity was
confirmed by paper chromatography in phenol
and in n-propanol-0.1 m ammonium acetate,
before and after acid hydrolysis. The imidazolyl
components at 4 hr. accounted for 38% of total C¥
activity of the extract; at 8 hr., 29.8%; and at 16
hr., 24.8%. Essentially all the C14 activity resided
in anserine. Creatine was isolated chroma-
tographicaily and accounted for 48.9% and 52.8%

Mar
of t
and
com
dire

102

Ss & Dm =a

80,

cap
rat
sys
is i
to

an¢
cho
cou
late
bot
phe
aci
in {
bio
ant
(ef.
sys
ph
lip’
ph
ph

inc
chl

ors
ing

ume 1§

rationg
60 min,
osomal
actions
or 3 hr,
amino
ontable
ytained
17: 111,
which
iar to
ons by
etween
1 these
tion of
tivities
nearly
tion of

mito-
it ap-
somes,
role in
somes

ne. I,
LLARD
Hosp.,
Yale

from
rabbit
, work
e rat.
intra-
ionine
8 hr.,
; were
114 in-
n the
ightly
were
y on
place
atine
zolyl
iuphed
0.2m
plish-
idine
was
henol
tate,
zolyl
a] CM
at 16
sided
oma-
2.8%

March 1956

of the total.activity at 4 and 8 hr., respectively.
Comparison of the specific activities of creatine
and anserine, assuming a similar half life for both
compounds, indicates that methionine acts as a
direct donor of the methyl group of anserine.

1020. Labeling of brain phospholipid in vitro.
W. C. McMurray, J. F. Berry anp K. P.
STRICKLAND (introduced by R. J. RossiTErR).
Dept. of Biochemistry, Univ. of Western Ontario,
London, Canada.

Previously (Proc. 3rd Internat. Cong. Biochem.
80, 1955) it was reported that there are 2 systems
capable of supporting phospholipid labeling in
rat brain homogenates: a) an anaerobic glycolytic
system, best observed in water homogenates, that
is insensitive to DNP and cyanide, but sensitive
to low concentrations of iodoacetate or fluoride
and b) an aerobic system, best observed in mito-
chondria from sucrose homogenates, that is un-
coupled by DNP, inhibited by cyanide, stimu-
lated by fluoride and insensitive to iodoacetate. In
both systems, some 90% of the activity of the
phospholipid could be attributed to phosphatidic
acid and diphosphoinositide, with lesser amounts
in the phosphatidyl choline (Dawson, Biochim. et
biophys. acta 14: 374, 1954). In both systems, ATP
and other acid-soluble phosphates were labeled
(ef. Federation Proc. 14: 183, 1955) and in both
systems biosynthetic ATP* and synthetic phos-
phorylcholine-P#2 were incorporated into the
lipid fraction without prior breakdown. The
phosphorylcholine-P#2 was present only in the
phosphatidyl choline. In both systems, CTP
(KENNEDY AND WelIss, J. Am. Chem. Soc. 77:
250, 1955) was found to be a cofactor for the
incorporation of inorganic P*? and phosphoryl-
chlorine-P*?. In the anaerobic system, added CoA
(5 X 10-5 M) increased the incorporation of in-
organic P%? into phospholipids, without affect-
ing the specific activities of the acid-soluble
phosphates.

1021. A colorimetric, micro method for acetyl-
cholinesterase. Don E. McOsKeEr* AND LOUISE
J. Danrev. Dept. of Biochemistry and Nutrition,
Cornell Univ., Ithaca, N.Y.

The need for a rapid, accurate and simple
method for the determination of acetylcholin-
esterase has been quite apparent. In the method
to be described, acetylthiocholine was used as
substrate; rat brain homogenate was the source
of enzyme; the production of sulfhydryl groups, as
determined by the nitroprusside reaction, served
as a measure of enzyme activity. Koelle (Proc.
Soc. Exper. Biol. & Med. 70: 617, 1949) used acetyl-
thiocholine as a substrate for acetylcholinesterase
in histochemical studies. He demonstrated that
the enzyme showed the same specificity for the

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

313

thioesters of choline as it did for the choline
esters. The possibility that the substrate was
hydrolyzed by enzymes other than acetylcholin-
esterase was eliminated by using eserine as an
inhibitor. Eserine (6 X 10-5 Mm, final concentra-
tion) was found to completely inhibit the hy-
drolysis. Optimum conditions were determined
by measuring the enzyme activity versus 1)
increasing substrate concentration, 2) increasing
enzyme concentration, and 8) time. Results
obtained by this method compare favorably with
results obtained using the Warburg manometric
technique. Quantities of enzyme which will
hydrolyze 0.125 um of substrate in 0.5 ml total
volume of incubation mixture per 10 min. have
been accurately measured using the colorimetric
procedure. This would correspond to about 3 ul
of carbon dioxide liberated in the Warburg method.
This method has the following advantages: it is
rapid, requires simple laboratory equipment, is
sensitive to very small amounts of enzyme and
the reaction is followed by the appearance of the
products of the reaction and not by the disap-
pearance of substrate.

1022. Methionine precursors in Neurospora.
Rosert A. McRorie anp GERALD L. CARLSON
(introduced by G. Howarp SATTERFIELD).
Dept. of Chemistry and College Agric. Exptl.
Station, Univ. of Georgia, Athens.

After growth for 1-2 wk. on unsupplemented
minimal media, mycelial extracts of a ‘leaky’
mutant of Neurospora crassa contain a compound
which supports growth of other ‘methionineless’
mutants of the same organism. The accumulated
compound contains an amino group and a thio
ether linkage which can be oxidized to an active
sulfoxide and an inactive sulfone as shown by
chemical and bioautographic tests on paper
chromatograms. Since the extracts support the
growth of certain Neurospora mutants which
respond to methionine but not to homocysteine,
the accumulated compound appears to be inter-
mediate between these compounds in methionine
biosynthesis. The chemical and biological proper-
ties of concentrates of the active extracts have
been shown to differ from those of the proposed
biosynthetic intermediates, S-hydroxymethyl
homocysteine and m-thiazane-4-carboxylic acid.

1023. Metabolic pathway of linolenic acid.
James F. Meap, GuntHer STEINBERG,* WIL-
LIAM H. SiLaToN, JR.* AND Davin R. Howron.*
Atomic Energy Project, Univ. of California
School of Medicine, Los Angeles.

In experiments designed to shed light on the
role of linolenic acid in essential fatty acid de-
ficiency, carboxy-labeled methyl linolenate was
administered to rats, and the lipids, fatty acids

314

and polybromo fatty acids were prepared from
their organs. Linolenic acid was found to be
readily converted to saturated acids and oleic
but not to linoleic. The ether-insoluble poly-
bromide fraction was debrominated, hydrogenated
and separated by chromatography into stearic,
arachidic and behenic acids. Degradation of the
arachidic acid in 4 one-carbon steps revealed that
its precursor was formed largely from linolenate
and acetate. However, the activity of the other
carbon atoms is not readily interpreted. The
evidence suggests that linoleic and _ linolenic
acids give rise in the body to 2 families of higher
unsaturated fatty acids which are not readily
interconvertible and have different physiological
actions.

1024. Increase of a liver enzyme in alloxan
diabetes and reversal by insulin. ALAN H.
Menter, E. G. McDanreLt* anp James M.
Hunpb.ey.* Natl. Insts. of Health, Bethesda, Md.
Urinary N-methyl] nicotinamide may be derived

from dietary niacin or from tryptophan. Con-

version of tryptophan to pyridine derivatives is
known to proceed via 3-hydroxyanthranilic acid.

The enzymatic conversion of 3-hydroxyanthranilic

to nicotinic acid or a niacin derivative has not

been reported, but enzymes are known: (I) that
oxidize 3-hydroxyanthranilic acid to an unstable
product, and (II) that convert the oxidation
product to picolinic acid (J. Biol. Chem. In press).
It has now been found that the levels of enzyme II
are increased several fold in the livers of alloxan-
diabetic rats, whereas the tryptophan oxidizing
system and 3-hydroxyanthranilic oxidase (I) are
not significantly changed. This finding provides
an enzymatic basis for the results of McDaniel,
Hundley and Sebrell, who reported that diabetic
rats excrete less N-methyl nicotinamide than
normal animals when given a test dose of trypto-
phan (Federation Proc. 14: 448, 1955). A similar
differegce. in N-methyl nicotinamide excretion
was found when 3-hydroxyanthranilic acid was
fed. These results suggest that in the diabetic
animals niacin synthesis is diminished by in-
creased diversion of a common metabolite through
an alternate (picolinic acid) pathway. Alloxan-
diabetic animals treated with insulin gradually
became capable of increased excretion of N-methyl]
nicotinamide, and the level of enzyme II in such
animals falls to the normal low values. Whereas
fasting has been found by various investigators to
produce changes similar to those caused by
alloxan in other liver enzymes, the level of en-
zyme II is not altered in the livers of fasted rats.

1025. Salt effects in the yeast hexokinase
system. JACKLYN B. MELCHIOR AND NorTEN C.
Metcuior.* Grad. School and Stritch School of
Medicine, Loyola Univ., Chicago, Ill.

FEDERATION PROCEEDINGS

Volume i

It has been demonstrated that polyphosphate
in solution form complexes with the mono- and
di-positive ions present. These complexes may be
important in explaining the influence of salts
upon the rate of reactions involving adenosine.
triphosphate (ATP). In the present investigation
the apparent activity of yeast hexokinase has been
measured in a variety of solutions either by
observing the glucose disappearance in 30 min. or
by following acid production colorimetrically. The
effect of altering the concentrations of K, Na, Mg
and tetramethylammonium (TMA) chlorides and
ATP was studied. The following phenomena
were observed as these were varied in turn: J)
increasing TMACI increases the rate of glucose
disappearance at low pus (about 7) but decreases
it at higher pus (8 and over). 2) Substitution of
Na* or Kt for TMA* under otherwise identical
conditions reduces the rate of the reaction. 8)
Increasing Mg increases the rate of the reaction by
an amount which depends upon the ATP con-
centration. 4) Increasing ATP concentration in-
creases the rate of H* production until ATP =
Mg; further increases are inhibitory.

1026. Biosynthesis of ergothioneine. Donaw
B. MELVILLE, STEPHEN Ercu* anp Martna I,
Lupwia.* Dept. of Biochemistry, Cornell Univ,
Med. College, New York City.

Ergothioneine, the betaine of thiolhistidine, has
been found in cultures of Neurospora crassa,
When the fungus is grown in a chemically defined
medium containing S**-sulfate, radioactive ergo-
thioneine is formed. Experiments with S*5- and
C'4-labeled compounds have been carried out to
study the pathway of biosynthesis. The carbon
and nitrogen skeleton of ergothioneine is ap-
parently derived from histidine, as evidenced by
the formation of radioactive ergothioneine when
N. crassa is grown in the presence of histidine-2-
C'*. Thiolhistidine, on the other hand, does not
serve as a precursor to any significant extent.
The sulfur of ergothioneine is provided by cystine
or methionine; either of these amino acids is more
effective than inorganic sulfate in serving as a
precursor for the sulfhydryl group. When the
fungus is grown in the presence of methyl-labeled
C'4-methionine, the ergothioneine which is pro-
duced contains radiocarbon, and this is present
almost exclusively in the methyl groups. (Aided
by grants from the Natl. Science Fndn. and Swift
and Co.)

1027. Mechanism of ATP activating effect on
brain AMP deaminase. J. MENDICINO (intro-
duced by Henry Z. SaBiE). Dept. of Biochem-
istry, Western Reserve Univ. Med. School, Cleve-
land, Ohio.

Muntz discovered in extracts of acetone-dried

dog brain an enzyme (J. Biol. Chem. 201: 211, 1958)

olume if

osphates
no- and
may be
of salts
enosine.
tigation
has been
ther by
) min. or
ly. The
Na, Mg
ides and
nomena
turn: 1)
glucose
ecreases
ution of
dentical
tion. 8)
ction by
CP con-
tion in-
ATP =

DoNAlD
RTHA L,
ll Univ,

ine, has
crassa,
defined
ye ergo-
335_- and
| out to
carbon
is ap-
need by
1e when
idine-2-
oes not
extent.
cystine
is more
ng as a
en the
labeled
is pro-
present
(Aided
d Swift

fect on

(intro-
tochem-
, Cleve-

\e-dried
1, 1958)

March 1956

which degminates AMP to IMP and NH. The
reaction proceeded only if stoichiometric amounts
of ATP were also present. This enzyme has now
been purified 20-fold and the nature of the ATP
effect has been investigated. Other nucleotides
(ITP, GTP, ADP, CTP) do not take part in the
reaction. Studies with AMP*? show that IMP
arises directly from AMP and that there is no
phosphate transfer between ATP and AMP.
Further interaction of AMP with ATP cannot be
demonstrated. No deamination occurs in the
absence of ATP and maximum rates are obtained
only in the presence of high levels of ATP (.007 m).
The enzyme cannot be activated by preincubation
with ATP and removal of the latter by dialysis.
It was considered that the reaction might proceed
as follows: (1) ATP— ITP + NH;; ITP + AMP @
ATP + IMP. However, when ITP is incubated
with AMP in the presence of the enzyme, no ATP
can be detected. It is possible that added ITP is
not comparable to that which might be produced
enzymatically. To test this, N15-ATP was pre-
pared by growing yeast in the presence of N1-
(NH,)2SO,. The acid soluble nucleotides were
extracted and purified. To examine the role of
ATP, N'5-ATP and unlabeled AMP, and N?-
AMP and unlabeled ATP are incubated with the
purified deaminase which has no myokinase
activity. These experiments, now in progress,
should show whether the 6-amino group of ATP
is involved in the reaction.

1028. Synthesis of the growth factor L-seryl-
L-histidyl-L-leucyl-L- valyl -L - glutamic
acid. R. B. MERRIFIELD AND D. W. Woo .tey.
The Rockefeller Inst., New York City.

A peptide possessing streptogenin activity was
recently isolated from a partial hydrolysate of
crystalline beef-insulin and its structure estab-
lished as serylhistidylleucylvalylglutamic acid. In
spite of the apparent purity of the isolated ma-
terial there remained the possibility that the
biological activity was due to trace amounts of a
very active impurity. For this reason, and because
larger amounts of the peptide were desirable for
other experiments, a synthesis of this penta-
peptide was devised. Carbobenzoxy-t-leucyl-t-
valine was prepared by the mixed anhydride
method and converted to diethylearbobenzoxy-t-
leucyl-L-valyl-L-glutamate by a similar procedure.
The tripeptide, diethyl-u-leucyl-t-valyl-t-gluta-
mate hydrochloride, was obtained by catalytic
reduction. Carbobenzoxy-t-seryl-.-histidine
methyl ester was made by the azide procedure and
purified by countercurrent distribution. This was
converted to the azide and condensed with the
above tripeptide ester to give diethyl carbo-
benzoxy-L-seryl-u-histidyl-t-leucyl-L-valyl-t-glu-
tamate in 54% yield. The desired pentapeptide
was obtained after removal of the protecting

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

315

groups with concentrated HCl. The overall yield
of pure peptide calculated from L-serine was 8%.
The synthetic peptide was indistinguishable from
the peptide previously isolated from insulin.
Strepogenin assay with Lactobacillus casei showed
an activity of 84 u/mg which was equivalent to
that of the natural material. This confirmed the
belief that the activity of the purified preparation
from insulin did indeed reside in the pentapeptide
serylhistidylleucylvalylglutamic acid.

1029. Bovine antifibrinolysin. Epwi1n T. Mertz,
RaupyH EILBERG* AND ARTHUR Da.tBy*. Dept.
of Biochemistry, Purdue Univ., Lafayette, Ind.
The filtrate from the 30% (NH,)2S0O, precipitate

used in the preparation of bovine fibrinolysin

(H. D. Jackson anp E. T. Merz, Proc. Soc.

Exper. Biol. & Med. 86: 827, 1954 contains sig-

nificant amounts of antifibrinolysin. By partition

of the proteins in the filtrate with (NH,)2SO,,
it has been possible to separate dialyzed fractions
which are about 1000 times more active per
milligram of nitrogen than the original dialyzed
filtrate solution. The filtrate and its fractions have
been assayed by determining their inhibitory
effects on bovine fibrinolysin. A new procedure
has been developed which makes use of the con-
ventional Warburg manometric technique. When

a fixed amount of fibrinolysin splits the ester

group of p-toluene-sulfonyl L-arginine methyl

ester, (TAME), the amount of CO: released from
bicarbonate buffer is measured. The quantity of

antifibrinolysin which reduces CO. output 50%

in this assay is used as a measure of inhibitor

activity. (Supported by a grant from the Surgeon

General’s Office, Dept. of the Army.)

1030. Reversible denaturation of trypsin by
urea. Harry O. Micuex,. Enzyme Chemistry
Branch, Chemical Corps Med. Labs. Army Chem-
ical Ctr, Md.

Trypsin has been reported to retain its activity
in 8.3 Mm urea even in the presence of diethyl-p-
nitrophenyl phosphate, although p-nitrophenol
is liberated (VISWANATHA AND LIENER, J. Biol.
Chem. 215: 777, 1955; Abstracts, American Chem-
ical Society, 128th Meeting, Minneapolis, Minn.,
34C, 1955). Our studies indicate that the esterase
activity of trypsin is lost in a few seconds when
measured in the presence of 8.3 M urea; complete
recovery of activity is obtained by diluting the
8.3 m urea solution of trypsin with 0.01 n HCl in
the absence of substrate and subsequently measur-
ing the esterase activity at pH 7.6 and at low urea
concentrations. In the presence of substrate and
5 or 6.7 m urea, loss of trypsin activity is first
order and proportional to a high power of the
urea concentration. In 8.3 mM urea and pu 7.6,
trypsin reacts with Sarin (isopropyl methyl phos-
phonofluoridate) at less than 1% of the rate of re-
action in the absence of urea. When one equivalent

316

of phosphorus is bound in the presence of urea,
about 75% of the esterase activity is still recover-
able. The results indicate that trypsin is rapidly
and reversibly denatured by high concentrations
of urea. The slow reaction between Sarin and
trypsin in the presence of urea may involve
tyrosine residues, in analogy with similar reactions
of Sarin with nonenzyme proteins (Discussions,
Faraday Society, 1955, In press).

1031. Transfer of the formyl group of form-
amidinoglutaric acid (FAG) to folic acid.
ALEXANDER MILLER (introduced by H. T.
CLARKE). Dept. of Biochemistry, Columbia
Univ., and New York State Psychiatric Inst.,
New York City.

The formation of N™® -formylfolic acid from
FAG and folic acid by a Dowex-2-treated, di-
alyzed, soluble protein fraction of calf liver re-
quires boiled liver or yeast extract. These extracts
are replaceable in part by reducing agents such as
sulfhydryl compounds and ascorbic acid or by
cyanide and full replacement of yeast extract is
obtained by further addition of citric acid, d-
isocitric acid or cis-aconitic acid in less than sub-
strate amounts. The addition of TPNH or treat-
ment of folic acid with NaBH, dispenses with the
need for tricarboxylic acid but not for reducing
agents. In the presence of reducing agents, di-
hydrofolic acid in catalytic amounts allows
complete formylation of 50 times its concentration
of folic acid. This finding suggests the presence of
a system catalyzing the transfer of hydrogen or of
the formyl group of reduced formylfolic acid to
folic acid. An enzyme system which specifically
catalyzes a reaction between FAG and tetra-
hydrofolic acid in the presence of a reducing agent
has been purified by ammonium sulfate fraction-
ation and negative adsorption. This reaction is
conveniently studied by measurement of a peak at
350 mu after acidification of the reaction mixture.
Spectroscepic and chromatographic evidence
indicate the formation, upon acidification, of
anhydrocitrovorum from N-formyltetrahydro-
folic acid or a closely related compound.

1032. Synthesis of amino acids under possible
primitive earth conditions. StTanitey L.
MILLER (introduced by G. L. Foster). Dept. of
Chemistry, California Inst. of Technology,
Pasadena, Calif.

Electric discharge in a mixture of methane,
ammonia, hydrogen and water has been shown to
yield amino and hydroxy acids and simple ali-
phatic acids (S. Mriuer, Science 117: 528, 1953;
J. Am. Chem. Soc., 77: 2351, 1955). This mixture
of gasses has been proposed as the composition of
the Earth’s atmosphere in the early stages of
formation. Examination of the system in greater

FEDERATION PROCEEDINGS

Volume 16

detail indicates that the synthesis of the amino
and hydroxy acids was accomplished by reaction
of the aldehydes and hydrogen cyanide produced
in the electric discharge to form amino and
hydroxy nitriles which were then hydrolyzed to
the corresponding acids. Several more amino acids
have been identified in the mixture of organic
compounds produced by long continued electric
discharges in the gas mixture. Using these results
as a guide, it is proposed that many of the organic
compounds that made up the first living organisms
were formed from reactions of the dilute solutions
of aldehydes, hydrogen cyanide and ammonia in
the primordial ocean. This mechanism of syn-
thesis does not require electric discharges to
operate; any system that forms aldehyde, hydro-
gen cyanide (and ammonia) will yield amino acids.

1033. Vitamin By» and uric acid synthesis in
the chick from C™ precursors. 8S. P. Mistry,
K. C. Tsene, anp P. B. RamaRao (introduced
by T. 8. Hamiiron). Div. of Animal Nutrition,
Univ. of Illinois, Urbana.

Since the chick is a convenient system to study
purine synthesis, the effect of vitamin By
deficiency on uric acid synthesis has been investi-
gated. Following the injection of sodium formate-
C4 and serine-3-C'* into normal and By-de-
ficient birds the specific activity of the uric acid
from 48-hr. urinary collections was found to be
approximately 1.6 times higher in the normal than
in the deficient birds. When glycine-2-C™ was
injected no difference in the S.A. of uric acid was
found. Following methionine-CH;-C! injection
and successive 4-hr. collections, in normal birds
the highest S.A. was found in the first 4-hr.
sample, whereas in the deficient birds the activity
reached a peak in the 8- to 12-hr. collection. This
maximum was only a third of that from the
normal chicks. Similar studies with formalde-
hyde-C'* gave a different picture. The highest
§.A. was found in the first 4-hr. collections from
normal as well as from deficient birds, and the
activity of the deficient peak was considerably
higher than that of the normal peak. This indi-
cates that vitamin By, functions in the biosyn-
thesis of the purine from one-carbon precursors,
formate, serine-8-carbon and methionine-methy]-
carbon, but it does not play a role in the synthesis
of uric acid from formaldehyde. Since glycine is
primarily incorporated per se, this would be ex-
pected to mask any Bi: effect on glycine utiliza-
tion as a one-carbon donor.

1034. In vivo and In vitro lipogenesis in the
irradiated rat. Margaret G. MoREHOUSE
AND Ronatp L. Searcy.* Univ. of Southern
California, Los Angeles.

The authors (Science 122: 158, 1955) have pre-

su)

the
USE
hern

pre-

March 1956

viously shown that irradiated rats exhibit a
marked stimulation in the in vivo incorporation
of acetate-1-C!4 into liver fatty acids and glycogen.
To ascertain the particular lipid fraction in which
increased lipogenesis occurs, C!4-acetate incor-
poration was studied in liver neutral fat and
phospholipids. In addition, similar fractions were
studied in the small intestinal wall. To eliminate
any variation due to irradiation induced anorexia,
comparisons were made between nonfasted rats,
48-hr. fasted and those given 1500 r prior to a
48-hr. fast. The animals were killed 2 or 4 hr. after
an intraperitoneal injection of sodium acetate-
1-C!4, In liver neutral fat from irradiated animals,
the radioactivity was increased 3-fold over that
of the nonfasted controls and 6-fold over that of
the fasted controls. Further, liver phospholipid
synthesis in the x-rayed rats was stimulated to
almost twice that of the fasted controls. In the
intestinal wall neutral fat comparisons of similar
lipogenesis showed little difference between
fasted x-rayed and normal rats. However, a 50%
reduction in labeled acetate incorporation into
the wall phospholipids was noted in irradiated
animals at both time intervals. This appears to be
related to previously observed reductions in total
phospholipid amounts occurring after similar
roentgen doses. In vitro studies by the authors
have supported the in vivo findings. It thus ap-
pears that the irradiation effect is manifest at a
cellular level. (Supported by Atomic Energy
Commission contract AT (11-1)-113, Project 6.)

1035. Quantitative cystine, cysteine and
methionine requirements of mammalian
tissues cultivated in vitro. JosepH F. Mor-
GAN AND HEeLen J. Morton.* Lab. of Hygiene,
Dept. of Natl. Health and Welfare, Ottawa,
Canada.

Chick embryonic heart fibroblasts cultivated
in vitro in a completely synthetic medium sur-
vived only a few days in the absence of sulfur-
containing amino acids. Under these conditions,
the tissues showed a specific requirement for L-
cystine, while p-cystine was completely inactive.
The cultures also showed a supplementary re-
quirement for methionine, which could be satis-
fied equally by either the L- or p-isomer. To ob-
tain normal survival of 30-40 days in these tissue
cultures, a careful balance between cystine and
methionine was required. L-cysteine was found to
replace the L-cystine requirement, but p-cysteine
was inactive. In the presence of L-cysteine, the
supplementary methionine requirement was filled
by either the t or p form. Various metabolic
products and derivatives of cysteine were tested
and found to be ineffective in replacing cystine,
with the possible exception of 8-mercaptoethyl-
amine. These observations indicate the com-

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS 317

plexity of the sulfur amino acid requirements of
tissue cultures.

1036. Isolation of lacteperoxidase. MartTIN
Morrison aND Howarp B. Hami.rTon (intro-
duced by Water R. Buoor). Dept. of Bio-
chemistry, Univ. of Rochester School of Medicine
and Dentistry, Rochester, N. Y.

A new procedure to isolate and purify the
enzyme lactoperoxidase has been developed. The
starting material, raw skim milk, is first treated
with rennin to coagulate the major protein,
casein. The soluble whey proteins which contain
all of the lactoperoxidase are then treated with the
ion exchange resin IRC 50 in the ammonium salt
form. Approximately 80% of the lactoperoxidase
is adsorbed in a batch treatment. The hemo-
protein is then eluted from the washed resin with
phosphate buffer. The eluent from the resin con-
tains the enzyme contaminated by 70% extraneous
protein, as judged by the absorption of the pro-
tein at 280 and 412 my. The enzyme is further
purified in the fraction precipitated between 2 and
4m (NH,)2SO,. The precipitate, now containing
60% extraneous protein, is redissolved in dilute
phosphate buffer po 6.0. The concentrated crude
enzyme solution is purified by column chromatog-
raphy employing the ion exchange resin IRC 50.
Three colored fractions are separated by this
process: 1) a fast-moving green lactoperoxidase
fraction I containing only a small percentage of
extraneous protein, 2) a more slowly moving
lactoperoxidase fraction II, and 3) the slowest
moving fraction which is a red-colored protein.
A similar procedure appears to be applicable to
mammalian tissue. Preliminary work on the
prosthetic group of the hemoprotein indicates that
the tetrapyrrole nucleus is bound to the protein
by covalent bonds. The porphyrin nucleus, as
judged by the absorption spectrum of its hemin,
seems to be neither protohemin, hematohemin nor
the hemins a.

1037. Identity of diisopropylfluorophospha-
tase and acylase. L. A. MounTer (intro-
duced by Stepan. Luprewie). Dept. of Bio-
chemistry, School of Medicine, Univ. of Virginia,
Charlottesville.

Enzymes (DFPases) which hydrolyze diiso-
propyl fluorophosphate (DFP) and related com-
pounds have been shown to have ubiquitous dis-
tribution but their activities in normal metabolism
have not been established. Available data show
that there is a similarity between the solubility,
stability to temperature, pH and ethanol and
between metal ion activation and pH optima of
hog kidney DFPase and Acylase, an enzyme which
hydrolyzes N-acyl amino acids. A comparison of
the rate and extent of the enzymatic hydrolysis of
DFP and N-acetyl-valine, -leucine, -methionine

318

and -alanine by a number of hog kidney fractions
indicated that a single enzyme was involved. Both
hydrolyses were similarly affected by a number of
noncompetitive inhibitors. Kinetic studies of pH
optima, hydrolysis of mixed substrates and with
competitive inhibitors also showed that 1 enzyme
hydrolyzed both groups of compounds. The
identity was further confirmed by paper electro-
phoresis studies. It is probable that the normal
function of DF Pase is associated with the hydroly-
sis or synthesis of amino acid derivatives.

1038. Biosynthesis of nucleic acid guanine:
the enzymic conversion of xanthosine-5’-
phosphate (XMP) to guanosine-5’-phos-
phate (GMP). H. S. Moyep* anv Boris
Maaasanik. Dept. of Bacteriology and Immu-
nology, Harvard Med. School, Boston, Mass.
Extracts of Aeobacter aerogenes, strain PD-1,

a purine-requiring mutant or of its parent strain,

1033, bring about the conversion of xanthosine-5’-

phosphate (XMP) to a guanine derivative. A

protein fraction obtained from sonically disrupted

cell suspensions of strain PD-1 by precipitation
with protamine sulfate catalyzed the following
reaction: XMP + ATP + NH,* — GMP + AMP

+ PP. Equimolar amounts of GMP, AMP and of

PP (pyrophosphate) were formed. AMP and GMP

were isolated from the deproteinized reaction mix-

ture by paper electrophoresis and _ identified

spectrophotometrically. GMP was also isolated by
chromatography on Dowex-2-formate and identi-
fied as guanosine-5’-phosphate by paper chroma-
tography in isobutyric acid-NH;. PP was isolated
by precipitation with MnSO, and estimated by

the method of Flynn et al. (J. Biol. Chem. 66:

791, 1954). In crude extracts glutamic acid, glu-

tamine or NH,* serves as amino donor. However,

in the aged purified protein fractions glutamate is
inactive, and glutamine is less active than NH,".

PP is formed by the purified fraction from ATP

only inathe presence of NH,* or glutamine and of

XMP. Hydroxylamine (8 X 10-* Mm) causes 65%

inhibition without the formation of hydroxamic

acid or of PP. Extracts of strain P-14, a guanine-
requiring mutant which excretes xanthosine and
is capable of synthesizing nucleic acid adenine
de novo failed to aminate XMP. The results indi-
cate that the dehydrogenation of IMP to XMP
and the amination of XMP to GMP are essential
steps in the biosynthesis of nucleic acid guanine.

1039. Rapid quantitative separations of glu-
tamic, aspartic and cysteic acids by ion-
exchange chromatography.! J. E. MuLDREY
AND Wiipa H. Martinez (introduced by W. B.
WENDEL). Southern Regional Research Lab.,
Southern Utilization Research Branch, Agric.
Research Service, U.S. Dept. of Agriculture,

FEDERATION PROCEEDINGS

Volume 1§
and Chemistry Dept., Loyola Univ., New

Orleans, La.

A method which includes a minimum of time,
a small number of ninhydrin determinations, and
a sample large enough to minimize analytical
error, has been developed for the separation of
glutamic, aspartic and cysteic acids. The amino
acids at a pH of 10-11 are adsorbed on a colum
of strong-base ion exchange resin in the acetate
form. A good separation of glutamic and aspartic
acids can be obtained by elution with 0.04 m ace-
tate buffer at a rate of 1-2 ml/min. By means of
stepwise increases in acetate molarity, a well
defined separation of glutamic, aspartic and
cysteic acids can be achieved in even less time,
Effluent is collected either manually, in fractions
of large volume, or automatically. Appropriate
fractions, selected via spot tests or experience,
are combined for photometric ninhdrin determina-
tions. Recoveries of the 3 acids have been con-
sistently 100+3%. Results are reported on the
application of the methods to cottonseed meal
and bovine serum albumin.

1040. Hemorrhagic kidneys in male rats of
different ages. Dwicut J. MULFORD AND
CHARLOTTE E. OuTLAND.* Dept. of Biochemis-
try, Univ. of Kansas, Lawrence.

Male rats (Sprague-Dawley) ranging in age from

3 wk. to 20 wk. were fed a low choline diet con-
sisting of casein 18%, yeast 6, agar 2, salt mixture
4, CaCO; 1, sucrose 48.7, l-cystine 0.3, lard 199
and Natola 0.1. Hemorrhagic degeneration did not
occur in the kidneys of these animals. When 2
amino-2-methyl-1-propanol was incorporated into
the diet above at levels of 10 and 20 mg/gm of
food, kidney hemorrhagic degeneration did occur
in at least 90% of all the animals in each age
group. The time of appearance of kidney hemor-
rhages varied with the starting age of the animals
as well as the level of 2-amino-2-methy]-1-propanol
in the diet. Animals 3, 4 and 5 wk. of age showed
hemorrhages in 4 to 7 days on 10 mg 2-amino-2-
methyl-1-propanol per gram of food while animals
12-20 wk. of age showed hemorrhages in 9 to l4
days on 20 mg 2-amino-2-methyl-1-propanol per
gram of food. Animals 12-20 wk. of age showed 4
much less severe hemorrhagic condition in the
same period of time on 10 mg 2-amino-2-methyl-l-
propanol. When choline chloride (5 mg/gm food)
was incorporated into the diet containing 2-amino-
2-methyl-1-propanol, none of the animals in any
of the groups showed hemorrhagic degeneration
in the kidneys.

1041. Titratable —SH groups of sickle-cell
hemoglobin at 0° and 38°C. Mak1o MuRAYAMA
(introduced by Cart C. NrEMANN). Gates and
Crellin Labs. of Chemistry, Calif. Inst. of Tech-
nology, Pasadena.

eo oC 8 0 oo =

=—a_ feo Ss oO fa st © %S @ DS Ss SP oO

re ®>

i iin n— . n Mie i, iii. a. i i i ee ee a ee |

o.;

lume 1§

» New

of time,
ns, and
alytical
ution of
e amino
columa
acetate
aspartic
1 M ace-
leans of
a well
tic and
33 time,
ractions
ropriate
erience,
ermina-
en con-
on the
d meal

rats of
2D AND
»chemis-

ge from
et con-
mixture
ird 199
did not
Vhen 2-
sed into
»/gm of
d occur
ich age
hemor-
animals
ropanol
showed
mino-2-
animals
9 to 4
nol per
owed &
in the
sthyl-l-
n food)
-amino-
in any
eration

le-cell
tAYAMA
tes and
f Tech-

March 1956

Harris (Proc. Soc. Exper. Biol & Med, 75: 197,
1950) found that a marked increase in the viscosity
of sickle-hemoglobin solution occurs upon de-
oxygenation. When the sickle-cell hemoglobin
concentration was greater than 10 gm/100 ml the
deoxygenation of the solution transformed it into
a semisolid gel-like state at 38°C. It was observed
in our laboratory that the semisolid gel-like de-
oxygenated sickle-cell hemoglobin solution lique-
fies when placed in an ice bath. The object of the
present investigation was to determine if there was
any relation of —SH groups to the provess of
sickle-cell hemoglobin molecular aggregation.
Amperometric argentometric titration of normal
and sickle-cell, deoxygenated hemoglobins gave
the following results: 4 —SH groups per molecule
were found for both hemoglobins at 0°C. At 38°C
only 2 —SH groups per sickle-cell hemoglobin
molecule and 3 —SH groups per normal hemo-
globin molecuie were titratable. The method used
was essentially the same as that of Ingram (Bio-
chem. J. 59: 653, 1955) who also reported 4 —SH
groups per molecule of normal human hemoglobin.

1042. In vitro precipitation of a plasma factor
by gelatin and heparin. Inwin M. Murray
(introduced by Epwarp Muntwyter). Dept.
of Anatomy, State Univ. of New York, Med.
College, Brooklyn.

Heparinized blood obtained from rats previously
injected with gelatin-stabilized radiogold colloid
resulted in precipitation of radiogold in the red
blood cell compartment following centrifugation.
Precipitation was prevented by injecting addi-
tional gelatin with the radiogold colloid followed
by recovery of the radiogold in the plasma com-
partment. ACD plasma, radiogold and heparin
were incubated for 20 mia., and centrifuged for
10 min. at 2700 rpm at 20°C. Aliquots of the super-
natant were counted by a scintillation counter
and compared with a standard. The amount of
radiogold precipitated was expressed as per cent
of total radiogold in the sample. Maximum pre-
cipitation of radiogold occurred with 100 ug of
gelatin and 15 ru of heparin/ml of plasma. In-
creased precipitation of radiogold occurred in
direct proportion to the amount of Cohn’s
Fraction III added to the plasma sample. Pro-
thrombin-free oxalated plasma did not inhibit the
precipitation of radiogold. Following incubation
of oxalated plasma at 37°C there was a decrease
with time in the amount of radiogold precipitated.
Paper electrophoresis studies with human plasma
demonstrated interaction of the gelatin-radiogold
with the alpha 2 globulins, whereas following
heparin treatment the radiogold remained at the
origin. This non-migrating radiogold component
is decreased in incubated oxalated plasma and
corresponds to the radiogold component that

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

319

precipitates in the test tube. It is suggested that
a labile factor in plasma complexing with gelatin
in the presence of heparin is precipitated from
plasma following centrifugation.

1043. Calcium and magnesium complexes of
ATP. Determination of dissociation con-
stants. L, NANNINGA (introduced by A. Szmnt-
Gyoreyi). Inst. for Muscle Research, Woods
Hold, Mass.

The Mg and Ca complexes of ATP were investi-
gated by means of an anion exchange resin accord-
ing to the method of Schubert (J. Phys. Chem. 56:
113, 1952). This method allows conclusions as to
stoichiometry and dissociation constant. Over the
pH range 4.5 to 9 several mixtures were prepared
for each pH, containing 7.5 ml veronal acetate or
trisbuffer, made to 0.1 m in Na by NaCl addition;
5 ml Na;sHATP or NasATP 5.10-? mM, made to
0.1 Min Na and set to the right pu; 0-10 ml MgCl:
5.10- in 0.1 m NaCl, 100 mg of the strong basic
Nalco-resin (very fine mesh) and with NaCl
0.1 m added to 25 ml. After shaking for 3 hr. at
room temperature the resin was centrifuged and
ATP plus MgATP were determined in the super-
natant by means of extinction at 260 mu. This is
allowed as the extinction of ATP at 260 mu does
not change upon MgCl, addition. In each case the
partition of ATP between resin and supernatant
is calculated: AR/(A — AM) = Ka, where A =
free ATP concentration; AR is ATP bound to
resin and AM is MgATP complex. AR = (Total
ATP — A — AM)-25/0.1. Therefore 1/Kg =
1/Ka® — M/k-Ka’,where ke = M-A/MA and K/
= K, for M = 0 (M = free mg concentration).
When 1/Kz is plotted against M straight lines are
obtained proving that Mg combines with ATP
1:1 for this pH-range. The slope gives 1/k, = ky,
the formation constant of this complex. For
MgATP ky is very dependent on pH: at pH 8 or
higher ky = 1800; at px 6.5 1450; at pu 5.5 700 and
at pH 5 and lower 400. For Ca ATP ky = 1100 at
pH 8 or higher, 800 at px 6.5; 480 at px 5.5 and 370
at pH 5 or lower. At pu 8 the value for MgATP
was confirmed by using cation exchange resin,
constant Mg and increasing ATP concentrations
and Mg determination. ATP* binds Mg about
1.5 times stronger than Ca, while Mg and Ca are
bound equally by ATPH*.

1044. Phosphate uptake in the erythrocytes
of heated human blood. RatpH Natvia anp
Opvar Skaue (introduced by Hans Hocn).
Surgical Research Labs., Med. College of Virginia,
Richmond.

Previous work indicates a deleterious effect of
heat, both in vivo and in vitro, on the glycolytic
activity of erythrocytes. In the present study P*
uptake during 4 hr. of incubation at 37°C has been

320 FEDERATION PROCEEDINGS

used as an indicator of the glycolytic activity of
human erythrocytes heated in vitro to 48°, 51°,
53° and 55°C for 3 min. The mean uptake in
erythrocytes from 35 normal individuals was
15.70%+0.98% of the P*? added to 1 ml blood/0.1
ml red blood cells. Heating for 3 min. at 48°C
caused no significant decrease in uptake of P*.
A significant drop in phosphate uptake was ob-
served in the erythrocytes of blood heated to
51°, 53° and 55°C, the uptake after 4 hr. amounting
to 92%, 89% and 80%, respectively, as compared
to the normal mean. These results add further
evidence to the existing data, indicating a detri-
mental effect of heat on the glycolytic activity of
red cells. A relationship is suggested between the
reduction in glycolytic activity and the previously
reported morphological changes, increased
fragility and hemolysis of red cells subjected
to heat.

1045. Iron-Linding activity of sugar-amino
acids. J. B. NEILANDS AND P. M. Towns.ey.*
Dept. of Biochemistry, Univ. of California,
Berkeley.

Under specified conditions, a strain of Micro-
coccus lysodeikticus has been found to require for
growth the addition to the culture medium of
various iron-binding substances. Fructose-phenyl-
alanine [N-(1’-carboxy)-phenylethyl-1-amino-1-
deoxy-p-fructose], prepared by the method of
Gottschalk (Biochem. J. 52: 455, 1952), also sup-
ported growth. Since these substances were found
by Borsook and his associates (J. Biol. Chem.
215: 111, 1955) to act with iron in stimulating
incorporation of amino acids in vitro into the pro-
teins of rabbit reticulocytes, the iron-binding
activity of fructose-phenylalanine was examined.
Electrometric titration of fructose-phenylalanine
gave a pKa’ value of 7.4; this buffer zone was
eliminated when the titration was performed in
the presence of ferric ion. It was concluded that
the sugdf-amino acids react with iron to form
stable chelate derivatives which in some respects
resemble the well known iron-ethylenediamine-
tetraacetic-acid chelate.

1046. Polyphenol oxidase in Piricularia
oryzae. Harotp A, NEUFELD, FRANcEs M.
LATTERELL AND RoBert L. WEINTRAUB (intro-
duced by Cart R. Brewer). Camp Detrick,
Frederick, Md.

Production of polyphenol oxidase activity has
been studied in 2 strains of the rice blast: fungus,
Piricularia oryzae, cultured in liquid media.
Homogenates prepared from acetone powders of
the mycelium of either strain are capable of oxidiz-
ing a variety of phenolic compounds including
catechol, hydroquinone, phloroglucinol, resorcinol
and p-cresol as well as o-phenylenediamine and

Volume 15

p-phenylenediamine. The enzyme occurs also in
soluble form in the culture medium. One strain,
which produces a black pigment, oxidizes phloro-
glucinol appreciably faster than catechol, hydro-
quinone or p-phenylenediamine. The other strain,
which is a mutant derived from the first, produces
a reddish-brown pigment and oxidizes catechol,
phloroglucinol, hydroquinone and p-phenylenedi-
amine at approximately equal rates. Both strains
oxidize resorcinol at a slower rate than the other
4 substrates. The ability of these preparations to
oxidize the meta di- and triphenols, resorcinol
and phloroglucinol, together with the ability to
oxidize monophenols distinguishes the Piricularia
enzyme from both tyrosinase and laccase. Work is
currently in progress to ascertain whether the
described activities are due to a mixture of laccase
and/or tyrosinase with a hitherto undescribed
enzyme or to a new type of polyphenol oxidase
which is able to oxidize a great variety of mono-
and polyphenols.

1047. Proteins of synovial fluid. Orro W.
NeEvHAUSs (introduced by ArtHur H. Smita),
Depts. of Anatomy and Physiological Chemistry,
Wayne Univ. College of Medicine, Detroit, Mich.
The proteins in the serum and synovial fluid of

individual cows are being studied in 2 ways, 1)

a comparison of the overall protein picture and

2) a comparison of individual proteins having

specific biological activity. Zone electrophoresis

on starch was used to separate the proteins,

Typical electrophoretic patterns were obtained

showing albumin plus alpha 1, alpha 2, beta and

gamma peaks both for sera and the corresponding
synovial fluids. With synovial fluid an albumin
plus alpha 1 region was greater than with serum

(51.5% vs. 42%) ; synovial fluid alpha 2 and gamma

regions were less than serum (10.0%, 25.5% vs.

12.0%, 33.5%); while the beta peaks were similar

for both (ca. 12%). To compare biologically active

proteins, the alkaline phosphatases of serum and
synovial fluid were studied. The enzyme activity
of the fluid is from 2 to 4 times as great as serum
on a volume basis, or from 20 to 30 times as great
per milligram of protein. After electrophoresis the
phosphatase peak for the sera was found to have
an Raip. of 0.410.04 while with synovial fluid
Raib. was 0.23+0.05. (Rap. is the distance from
the origin divided by the distance of the albumin
peak from the origin.) No conclusive evidence was
obtained to show that this difference was the
result of complex formation between enzyme and
some serum protein not found in synovial fluid.

These data therefore strongly suggest that the

alkaline phosphatase of the bovine synovial fluid

and of the serum are dissimilar entities.

1048. Nature of 24-dehydroergosterol. H. J.
Nicuoxas, H. J. BurHuer, L. J. DAcKsEL AND

me 15

so in
strain,
hloro-
1ydro-
strain,
»duces
echol,
enedi-
trains
other
ons to
reinol
ity to
ularia
Tork is
ar the
aAccase
cribed
xidase
mono-

o W.
MITH),
nistry,
Mich.
uid of
ys, 1)
e and
aving
oresis
teins,
;ained
a and
nding
yumMin
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amma
To V8.
imilar
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tivity
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great
is the
have
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from
yumin
e was
s the
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t the
fluid

H. J.
. AND

March 1956

W. P. Young (introduced by James ALLIson).
Central Research Dept., Anheuser-Busch, Inc.,
St. Louis, Mo.

When yeast is grown in a well-aerated medium
and then introduced to a postgrowth period
utilizing only carbohydrate and phosphate, the
ergosterol content is increased (Chem. Abstracts
45, 3904c). The nonsaponifiables from such yeast
exhibit the characteristic ultraviolet absorption
peaks of ergosterol plus a rather intense peak at
230 mu. Preparation of ergosterol from the non-
saponifiables gives a product which has the proper-
ties of 24(28)-dehydroergosterol (J. Org. Chem.
19: 1734, 1954). In our hands acetylation of the
crude nonsaponifiables and repeated crystalliza-
tion yields ergosterol acetate plus a gummy frac-
tion exhibiting absorption in the ultraviolet at
230 my» only. By fractional crystallization the
gummy material yields zymosterol and a crystal-
line sterol exhibiting only end absorption in the
ultraviolet. This sterol does not appear to be
identical to any of the minor yeast sterols reported
in the literature. After removal of all solids from
the gummy residue by crystallization, chromatog-
raphy of the still amorphous material on deacti-
vated alumina yields 2 fractions, 1 absorbing
intensely at 230 my and a 2nd absorbing with about
one-third the intensity of the former. Because of
the questionable purity of these fractions (both
amorphous) their nature remains unknown. The
nonsaponifiable fraction from yeast prepared in
the manner described appears to contain several
minor substances foreign to yeast grown in the
usual manner. A substance having the properties
of ‘24-dehydroergosterol’ was resolved into
ergosterol and several of these minor constituents.

1049. Hydration of pyridine as a determinant
in specificity. ALFRED NisoNnoFrr* AND Davip
PressMAN. Roswell Park Memorial Inst.,
Buffalo, N. Y.

Since biological reactions usually take place
in aqueous solution, the extent of hydration of
molecules involved may be an important factor in
determining specificity. In an earlier investiga-
tion, it was found that pyridine derivatives
combine with antibody homologous to the
p-azobenzoate ion in such a manner as to indicate
steric effects corresponding to the presence of a
large group attached to the annular nitrogen atom.
This was interpreted as indicating the presence
of water of hydration of the nitrogen atom. A
similar steric effect has now been observed in the
combination of pyridine derivatives with antibody
specific for another haptenic grouping, the p-(p’-
azophenylazo)-benzoate ion. Also, evidence for
the participation of water of hydration in a sero-
logical reaction has been obtained by another
experimental approach, in which antibody was

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS 321

prepared against the 3-azopyridine group. By
measurement of the extent of combination of vari-
ous haptens with the antibody, it was found that
the antibody is able to accommodate a large
group in the region complementary to the annular
nitrogen atom. This indicates that the antibody is
formed against the hydrated form of pyridine.

1050. Crystallization of myokinase. LAFAYETTE
Nopa AND STEPHEN A. Kusy.* Inst. for Enzyme
Research, Univ. of Wisconsin, Madison.

By a modification of procedures previously re-
ported (Federation Proc. 14: 261, 1955), myokinase
from rabbit skeletal muscle has been obtained as
needles from ammonium sulfate and as rhombo-
hedrons when a small amount of ADP was also
added. The fractionation procedure presently
being used consists of the following steps: a) acid
denaturation of inert proteins at px 2; b) precipi-
tation of zinc-proteinates at pH 7; c) fractional
extraction of the zinc-proteinates with 0.25 m
ammonium citrate at pH 8; d) ammonium sulfate
fractionation between 58-88% saturation at pH
8; e) precipitation by 0.0005 m AgNO; and 60%
saturated ammonium sulfate; f) adsorption on
IRC-50 (XE-64) resin at px 7.6 and 0.005 m glycyl-
glycine followed by elution with ammonium
citrate. The preparation represents a 160 to 200-
fold purification over the initial extract in 25-30%
yield. Crystallization has been carried out near
0° from about 1.5% solution at px 6.05 and about
62% saturated ammonium sulfate. In our assay
system using ATP-creatine transphosphorylase
(conditions which may not be optimal), velocities
of 110,000 m ATP formed per 100,000 gm protein
or about 23,000 m ATP per mole of myokinase have
been measured. This is comparable to a ‘turnover
number’ of 210,000 based on ADP reported. by
Bowen and Kerwin (Arch. Biochem. & Biophys.
57: 522, 1955). The preparation shows a single
sedimenting component and essentially single
moving boundaries in the Tiselius (pH 5.5-8.2).
The isoelectric point is about 6.05. The molecular
weight is 21,000 calculated from S,;° 2.39 X 107!%
and D,,;° 0.102 X 10-* (both measured in 0.15 m
KCI-0.01 m pu 7.0 PO,) and assuming V 0.73.

1051. Oxidative phosphorylation of isolated
yeast granules. Peter M. Nossau,* Bruce
Keecu* anp Merton F. Urrer. Depts. of Bio-
chemistry, Univ. of Adeiaide, Adelaide, Australia,
and Western Reserve Univ. Med. School, Cleveland,
Ohio.

The washed particulate fraction (granules) ob-
tained from yeast extracts prepared by 10-sec.
high-speed mechanical disintegration oxidizes
succinate, ethanol, isocitrate, a-ketoglutarate
and lactate when the appropriate cofactors are
added. In the presence of ATP, glucose and hexo-
kinase, the oxidation of all of these substrates is

322

accompanied by disappearance of inorganic phos-
phate. The phosphorylations, in most cases with
P/O values between 0.5 and 1.0, are completely
uncoupled by 2,4-dinitrophenol. In the case of
succinate oxidation, the P/O values can be raised
by washing and suspending the granules in 20%
sucrose or bovine serum albumin rather than in
salt solutions. Versene and Mg ions are required,
although Mn ions can replace the latter partially.
ADP but not AMP can replace the hexokinase
trapping system. The P/O values obtained are
greatly influenced by the duration of disintegra-
tion. With 5-sec. periods, the P/O values with
succinate may be raised to well above 1. Con-
versely, with 30-sec. periods, phosphorylative
activity is greatly reduced although oxidative
activity remains. The supernatant fraction of
such yeast extracts contains a factor(s) which can
completely uncouple the phosphorylation associ-
ated with succinate oxidation. The factor is heat-
labile, nondialyzable against NaCl-versene,
nonsedimentable at 110,000 g for 60 min., and does
not dephosphorylate ATP or HMP. The nature
and properties of this inhibitory factor are under
investigation. Preliminary indications have been
obtained that supernatants may also contain a
factor which enhances phosphorylation. (Sup-
ported in part by the Rockefeller Fndn. and by
the Atomic Energy Commission, Contract No.
AT (30-1)-1050.)

1052. Mitochondria isolated from polyvinyl-
pyrollidone-sucrose homogenates of rat
liver. ALEx B. Novixorr. Dept. of Pathology,
Albert Einstein College of Medicine, New York
City.

The morphologic and biochemical integrity of
rat liver mitochondria is excellently preserved
when isolated from PVP-sucrose homogenates.
(Homogenization is done in a mixture of 7.3%
PVP and 0.25 m sucrose, brought to pu 7.6-7.8
with alkali so that the resulting homogenate is
pH 6.9-7.1.) The mitochondria retain their typical
elongate form (phase-contrast microscopy) and
fine structure (electron microscopy) much better
than those isolated from sucrose alone. Their
‘ATP-ase’ activity is low and can be increased
by ageing or dinitrophenol, and they are capable
of oxidative phosphorylation without fluoride.
Repeated washing of the isolated PVP-sucrose
mitochondrial fraction reduces its esterase ac-
tivity and RNA content in parallel fashion, in-
dicating that the RNA, like the esterase activity,
is due to microsomal contamination. If the mito-
chondria contain any RNA, it is too little to be
demonstrable by our methods. When PVP-sucrose
mitochondria are observed under a coverslip, they
do not balloon into the large spheres containing a
dark crescent-shaped area at one pole. Such

FEDERATION PROCEEDINGS

Volume 1

swelling is achieved by addition of either distilled
water or desoxycholate. Electron micrographs
show the inner mitochondrial membrane
(‘cristae’) localized in the crescent-shaped areas,
Higher desoxycholate concentrations cause com-
plete disintegration of the mitochondria.

1053. Examination of acid-soluble fraction of
E. coli: comparison between normal and
bacteriophage-infected cells. James fF,
O’DONNELL AND R. P. Mackat (introduced by
J. J. CertHaMu). Dept. of Biochemistry, Univ, of
Chicago, Chicago, Ill.

A study has been made of the components of
the acid-soluble portion of E. coli and compared
with cells infected with the virus Ter. Cells of
Escherichia coli, strain B, were harvested in the
log phase of growth, centrifuged in the cold and
extracted with cold 3.6% perchloric acid. In the
infected system the cells in the log phase were
centrifuged, resuspended in } the original volume
of fresh media and infected with Ter. After aera-
tion for 14 min. the cells were immediately chilled,
centrifuged and extracted with 3.6% perchloric
acid. The extracts were prepared and chromato-
gramed on Dowex-1l according to Potter et al.
(J. Biol. Chem. 209: 23, 1954). Six milliliter frac-
tions of eluate were collected and read in a
Beckman spectrophotometer at wavelengths of
260 and 275 my. Individual peaks were taken to
dryness and rechromatogramed on Dowex-l.
Peaks from the rechromatograms were identified
by acid and base spectra and paper chromatog-
raphy. The solvent systems for the latter were
isobutyric acid-ammonium hydroxide and buffered
ammonium sulfate-n-propanol. In the normal
system the following compounds have been identi-
fied: in peak I, uracil and adenosine; peak II,
adenine, hypoxanthine, inosine, xanthosine,
CMP and AMP; peak III, a guanine compound;
peak IV, a uracil compound; peak V, a uracil
compound; peaks VI and VII, unidentified. In
the infected system, peak I of the normal system
is missing and there are 2 new peaks which did not
appear in the normal system. The identification
of these peaks is in progress at the present.

1054. Adverse effect of ascorbic acid on
stability of epinephrine and norepineph-
rine in acid solution. M. JANE OESTERLING
(introduced by Marion Fay). Woman’s Med.
College of Pennsylvania, Philadelphia.
Although ascorbic acid retards the atmospheric

oxidation of epinephrine and _ norepinephrine

which occurs readily in neutral or alkaline solu-
tions, it is here found to increase the rate of de-
struction of these adrenal amines in acetate-
buffered solutions of acid px. In a typical
experiment at px 5 the percentage of norepineph-

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March 1956

rine (original concentration: 3 X 10-‘m) remaining
after 5 days at room temperature was 99.2; with
added copper (10-5 m), 97.0; with ascorbic acid
(10-8 m), 67.1, and with ascorbic acid plus copper,
57.4. For epinephrine the values were 88.7, 74.7,
52.2, and 35.8, respectively. Potassium thiocyanate
partially suppressed the destructive effect of
ascorbic acid, presumably by removing cupric
ions from solution to a large extent. Cupric ions
promote the destructive effect of ascorbic acid
probably by catalyzing formation of dehydro-
ascorbic acid. Since the latter is very labile at
px 7 or above, especially in the presence of phos-
phate, but relatively stable at px 5 or below in
the absence of phosphate, it is suggested that this
marked difference in stability offers an explanation
for the divergent effects of ascorbic acid on ad-
renal amines at these different pH values. Addition
of dehydroascorbic acid directly to solutions of
adrenal amines caused a destructive effect similar
to, but somewhat less pronounced than, the
ascorbic acid effect at px 5. It caused no significant
destruction at pH 4. Consequently, the adverse
effect of ascorbic acid can be attributed only par-
tially to the formation of dehydroascorbic acid.

1055. Gelatinase action of Cl. histolyticum,
James D. OaiE* anp Mian A. Loaan. Dept.
of Biological Chemistry, Univ. of Cincinnati
College of Medicine, Cincinnati, Ohio.

Cl. histolyticum produces several extracellular
enzymes involved in the conversion of gelatin
into small peptides and amino acids. At least 2
of these enzymes are metal containing peptidases
and can be completely inactivated with versene.
By alcohol precipitation, an enzyme has been
obtained which liquefies gelatin and dissolves
denatured collagen with the formation of products
which are mainly nondialyzable. N-terminal group
analysis of these products by the DNP method
showed that glycine was the only detectable
N-terminal group uncovered by action of the
enzyme.

1056. Conversion of lanosterol to cholesterol.
James A. Otson* aNp Konrap Btiocu. Dept.
of Chemistry, Harvard Univ., Cambridge, Mass.
Lanosterol (4,4’,14 trimethyl cholestan-8,24

diene-38-ol) is readily converted to cholesterol by

rat liver homogenates. The pathway for this trans-
formation, however, is not known. The probable
first reaction in each of the demethylation proc-
esses is oxidation to a primary alcohol, followed
either 7) by release or transfer of formaldehyde
or 2) by further oxidation to an acid with subse-
quent decarboxylation. Lanosterol, labeled in the

4,4’, and 14 methyl substituents, was prepared

biosynthetically by injecting rats interperi-

toneally with 2-C™ sodium acetate. When this

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

323

lanosterol was incubated with rat liver homo-
genate in the presence of semicarbazide, choles-
terol was formed, but neither the trapped
formaldehyde nor the amino acid fraction (serine)
was labeled. On the other hand, either in the
presence or absence of semicarbazide, significant
radioactivity appeared in the CO:, and for every
mole of cholesterol formed, roughly 3 m of CO;
were produced. Very little CO. was formed dur-
ing incubation with lanosterol prepared bio-
synthetically from 1-C™ acetate, or with
cholesterol (from 2-C™ acetate). Small amounts
of a relatively polar chromatographic fraction
(‘diol’ group), steroid acids, and several other
fractions accumulated during the transformation.
Similar chromatographic fractions isolated from
rat injection experiments were converted to
cholesterol and COz in good yield. These results
suggest that in the conversion of lanosterol to
cholesterol, methyl groups are removed by the
general mechanism: R—CH; — RCH:OH —
RCOOH — RH + CO:.

1057. Chemical composition of cerebral
tissue. Norman 8. OLsen anp Doris H.
Criovert.* Research Lab.,VA Hosp., Nashville,
Tenn.

Although the chemical composition of brain
has been studied by many workers, the simultane-
ous analyses of labile and stable constituents
have not been reported in the same animal. In the
present study dogs were anesthetized with vinyl
ether and the skull and femoral blood vessels
were exposed. The animals were immobilized with
dihydro-8-erythroidine hydrobromide and main-
tained with artificial respiration. Electro-
encephalograms were obtained by skull electrodes.
The brain was frozen by pouring liquid air on the
exposed skull. Samples of arterial plasma were
obtained prior to freezing and analyzed for
glucose, inorganic phosphate, lactic and pyruvic
acids to ascertain proper respiration. The frozen
brain tissue was separated into gray (cerebral
cortex) and white (inner) matter, and dry weight
determined. Aliquots were analyzed for carbo-
hydrate intermediates, lipid fractions and free
amino acids. The present series consists of 12
normal dogs. The following are mean values ob-
tained for gray matter: dry weight 23.0%; glucose
4.0, glycogen 6.6, lactic acid 2.1, pyruvic acid
0.20, phosphocreatine 2.0, ATP 1.5, ADP 0.9, and
inorganic phosphate 3.0 mm per 1000 gm, respec-
tively; total lipids 54.4, total phospholipids 27.8,
alkaline hydrolyzable phospholipids 16.5, total
choline 12.9, cerebrosides 1.7, cholesterol 10.6,
phosphatidylserine 3.9, phosphatidylethanol-
amine 7.9, phosphatidylcholine 4.8% of dry
weight. The values for the same constituents in
white matter show a similar pattern.

324

1058. Effect of insulin and alloxan diabetes
on glucose transport into cells of perfused
rat heart. C. R. Park, L. H. JoHnson* anp
Joun H. Wricut, Jr.* Dept. of Physiology,
Vanderbilt Univ. Med. School, Nashville, Tenn.
Fifteen milliliters of medium containing 150

mg% glucose, 100 mg% sorbitol-1-C“ and 5%
albumin were recirculated through the isolated rat
heart (BLEEHAN AND FisHEr. J. Physiol. 123: 260).
The addition of 0.3 ug/ml insulin caused a 2-3x
increase in glucose utilization by the fasted normal
heart. This effect was much smaller in the alloxan
diabetic heart. The medium to muscle concentra-
tion ratios for sorbitol and glucose were estimated.
The sorbitol ratio was found to be about 0.25. It
was not significantly increased by insulin or the
duration of the perfusion. Since sorbitol was not
metabolized, this ratio therefore provided an
estimate of the extracellular space. In the absence
of insulin, the glucose ratio indicated a space that
was slightly less than extracellular in the normal
and diabetic muscle. With insulin, the glucose
space was increased 2-3x, indicating a considerable
accumulation of free intracellular sugar. The
intracellular glucose was appreciably less in the
diabetic. When the nonmetabolizable pentose
L-arabinose was perfused in place of glucose, the
sugar did not enter the cells of the normal or
diabetic heart, but penetrated in the presence of
insulin. These observations support earlier evi-
dence (Park et al. Am. J. Physiol. 182: 12, 17) that
insulin accelerates glucose transport through the
cell membrane of muscle, a process that antecedes
glucose phosphorylation and is rate limiting for
glucose utilization. It is further concluded that
the inhibition of glucose utilization in alloxan
diabetic muscle is due to inhibition of the trans-
port process.

1059. Apparent synthesis of carbohydrate
from fat in developing Ascaris eggs. RICHARD
F. Pagsey* anp Donatp Farrparrn. Inst. of
Parasitology, McGill Univ., Macdonald College,
Quebec, Canada.

Fertilized eggs of the pig nematode, Ascaris
lumbricoides, contain large reserves of glycogen,
an unidentified glucose-containing carbohydrate
and triglycerides. In normal development the eggs
are permeable only to gases. During development
to the vermiform stage, triglycerides and carbo-
hydrates decrease, the latter to about } the initial
amount. Subsequently the embryo molts once
before becoming infective and both glycogen and
nonglycogen carbohydrates are restored to their
original levels, whereas triglycerides decrease
faster than before. Protein and nonprotein
nitrogen remain essentially constant. Changes in
the carbon found in the glycerol moiety of the
triglycerides, ‘glucogenic’ volatile fatty acids, and

FEDERATION PROCEEDINGS

Volume 1§

water- and ether-soluble organic acids are in-
sufficient to account for the increase in carbo-
hydrates. Acid-soluble phosphorus compounds

are not abundant. Carbon dioxide is fixed into |

glycogen and nonglycogen carbohydrates, but
preliminary experiments indicate that this fixation
does not contribute greatly to net synthesis. All
results to be reported support the view that in
ascaris eggs fat is used for synthesis of carbo-
hydrate.

1060. Incorporation of adenosine-5’-phos-
phate into ribonucleic acid (RNA) by tumor
homogenates. A. R. P. Paterson* Anp G. A,
LePace. McArdle Memorial Lab., University
of Wisconsin, Madison.

Edmonds and LePage reported that incorpora-
tion of glycine-2-C* into mononucleotide purines
occurs in the soluble fraction of Flexner-Jobling
carcinoma homogenates (Federation Proc. 13:
202, 1954). A small incorporation of radioactivity
into RNA purines was obtained in similar experi-
ments by LePage (Cancer Research 13: 178, 1953).
The present study used the same system of gly-
colyzing tumor homogenate to measure the in-
corporation into RNA of adenosine-5’-phosphate
labeled with radiocarbon. Whole homogenates
were compared with homogenates from which
nuclei had been removed by centrifugation.
Lysing the nuclei and adding them back to the
cytoplasmic fraction increased by more than
twice the incorporation obtained with cytoplasm
and gave results superior to those with whole
homogenate. This can be interpreted to mean
either that the nuclei possess enzyme activity,
which is prevented from acting by a permeability
barrier, or that lysed nuclei contribute phospha-
tase activity which leads to a nucleotide poly-
phosphate balance more favorable to synthesis,
Experiments are being conducted to determine
which of these interpretations is correct.

1061. Localization, partial purification and
characterization of some aminopeptidases
from ascites tumors. EvizaABETH K. PaTTER-
SON AND EsTELLE PopBER (introduced by
Gerrit TorennisEs). Inst. for Cancer Research
and Lankenau Hosp. Research Inst., Phila-
delphia, Pa.

Binkley, Olson and Torres (Abstr. Am. Chem.
Soc. 36C, Minneapolis, Sept. 1955) reported that
hog kidney aminopeptidases are nucleoside di-
phosphate glucosamine peptide complexes.
Confirmation of this finding is being attempted
using Lettré-Ehrlich mouse ascites tumor cells
have a similar content of leucineaminopeptidase
per mg N as reported for hog kidney. Ultra-
microchemical methods were used for enzyme
assay and protein N determinations. Peptidases

plume 1§

are in-
1 carbo-
npounds
ced into
es, but
fixation
esis. All
that in
’ carbo-

'-phos-
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DG. A,
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orpora-
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Jobling
oc. 13:
uctivity
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, 1953),
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gation.
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oplasm
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- mean
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ability
ospha-
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ermine

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.\ TTER-
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tidase
Ultra-
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idases

M arch 1956

hydrolyzing glycyl-t-leucine (GL) and L-leucine-
amide (LA)* were found to be localized 95-100%
in the supernatant (Spinco L, 105,000 g) from
homogenates in buffered 0.9% NaCl, 0.25 m sucrose
or polyvinylpyrrolidone-sucrose. Experiments on
ultracentrifugal fractions, prepared by S. Sorof,
showed the peptidase activity to be associated
with proteins of relatively low molecular weight.
When the fractionation procedure of Binkley
was followed, the specific activity towards GL
increased 2-fold after chymotrypsin-trypsin di-
gestion, but decreased markedly on alcohol pre-
cipitation, confirming the lability of this amino-
peptidase. However, the more stable La-peptidase
retained 25% of the original activity after this
procedure plus chloroform-octanol shaking (Ci
13.4, 0.002% protein). Treatment of the alcohol
precipitate with ribonuclease followed by repre-
cipitation with alcohol gave a recovery of 30% with
800-fold total increase in specific activity (Ci
25.2, 0.001% protein). Ultraviolet absorption
curves of this material were closest to those cal-
culated for guanine-uridine dinucleotides. Further
reduction of the protein concentration by chloro-
form-octanol resulted in almost total loss of
LA-peptidase activity while the U-V spectrum
showed no significant change.

1062. Enzymatic degradation of heparin.
A. Natt Payza AND Epwarp D. Korn (intro-
duced by Grorce H. Hocesoom). Nail. Insts.
of Health, PHS, Bethesda, Md.

A flavobacterium has been described previously
(A. N. Payza anp E. D. Korn, Nature. In press)
which is able to use heparin as its sole source of
carbon. The bacteria can be grown in large quan-
tity on a nonheparin medium and then be adapted
to heparin utilization under nongrowing condi-
tions. Aqueous extracts of acetone-dried adapted
cells (but not of nonadapted cells) are able to
catalyze the hydrolysis of heparin (0.3 mg of
enzyme protein/mg of sodium heparin) at such a
rate that all metachromatic activity disappears
in 1 hr. The optimum pu for this reaction is 7.5
to 8.0, it is inhibited in the presence of 0.2 m salt
and the enzyme is inactivated by heating for 5
min. at 40°. Upon incubation of 1 mg of sodium
heparin (117 u/mg; gift of L. L. Coleman, The
Upjohn Co.) with an excess of enzyme (0.8 mg of
protein), 1.4 to 1.8 um of reducing group were
formed, no free amino group was produced (nin-
hydrin reaction) and an increase in periodate con-
sumption equivalent to 6.1 uM/ym of reducing
group formed occurred. Longer incubation, even
with the addition of more enzyme, produced no
further change. These results indicate that the
extract contains a glycosidase (which, assuming
the heparin to be at least 80% pure, splits heparin

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

325

to disaccharides) and an alcohol sulfatase but no
amino sulfatase.

1063. Enzymatic synthesis of an alpha 1,3’-
linked glucosyl disaccharide. Joun H.
Pazur, Tania Bupovicu* AnD Cari TipTon.*
Dept. of Biochemistry and Nutrition, Univ. of
Nebraska, Lincoln.

It was noted previously that a reducing com-
pound with an Rf value intermediate between
glucose and maltose was produced during the
conversion of maltose into a series of alpha 1,6’-
linked oligosaccharides by the transferring enzyme
of Aspergillus oryzae (PazuR AND FReEncuH. J.
Biol. Chem. 196: 265, 1952). This compound has
now been isolated in pure form and has been
characterized structurally. The specific rotation
of the compound is +89°. It reduces alkaline cop-
per sulfate but does not react with resorcinol.
Partial acid hydrolysis of the oligosaccharide
yields glucose and unhydrolyzed compound while
extensive acid hydrolysis converts the oligo-
saccharide quantitatively to glucose. The crystal-
line phenylosazone derivative of the compound
possesses the same melting point as turanose
phenylosazone and yields an x-ray diffraction
pattern identical to that of turanose phenylo-
sazone. The presence of an alpha 1,3’-glucosidic
bond in the oligosaccharide is established and
the constitution of the oligosaccharide is ap-
parently 3-O-a-p-glucopyranosyl-p-glucose. The
transferring enzyme of A. oryzae is thus capable
of effecting a transfer of glucosyl moieties to the
3 position of cosubstrate glucose molecules as
well as to the 6 position. This type of action
pattern may exist for other transferring enzymes
involved in the synthesis of carbohydrates and
may account for the presence of structural irregu-
larities in polysaccharides of the starch, dextran
and pentosan types.

1064. Role of iron, molybdenum and flavin in
activity of Clostridium butylicum hydro-
genase. Harry D. Peck, Jr.* anp Howarp
Gest. Dept. of Microbiology, School of Medicine,
Western Reserve Univ., Cleveland, Ohio.

The cell-free hydrogenase of Clostridium
butylicum is unusual in that it shows virtually no
activity when tested by reduction of methylene
blue and similar acceptors with H:. Ephemeral
activity can be demonstrated only if a low redox
potential is established, but such conditions are
not easily achieved in the ordinary type of dye
reduction assay. The enzyme can, however, be
readily measured by the ‘evolution assay’ (Bact.
Proc. p. 117, 1955) which is based on formation of
Hz from reduced methyl viologen, a low redox
potential dye. This assay was used to follow purifi-
cation of the soluble hydrogenase, which was en-
riched 60-fold to a specific activity of 2.5 million

326

(QH2(N) at 30°C). The resulting colorless prepa-
ration (1-2 mg protein/ml) was remarkably stable
to oxygen and freezing. Marked stimulation of
‘evolution activity’ by Fe**, MoO; and flavin
could be demonstrated after extensive dialysis of
enzyme treated with o-phenanthroline. Hydrogen
evolution by the purified enzyme was completely
inhibited by 100% CO; this provides evidence
that the photochemical action spectrum obtained
by Kempner and Kubowitz (Biochem. Zischr.
265: 245, 1933) for H: formation from pyruvate
by CO-inhibited intact clostridia is that of the
ferrous hydrogenase enzyme. The available data
are consistent with the mechanism advanced by
Peck et al. (Proc. Nat. Acad. Sc., Jan. 1956) and
suggest that ferrous iron is a component of the
site involved in primary activation (or terminal
formation) of Hz, while Mo and flavin participate
in electron transfer from (and to) this site.

1065. Relation of dietary cholesterol to es-
sential fatty acid deficiency. James J.
PreIFER* AND Rautpu T. Hotman. Hormel Inst.,
Univ. of Minnesota, Austin.

Previous work (Arch. Biochem. & Biophys. 57:
520, 1955) has demonstrated that conditions
which promote hypercholesterolemia, i.e. diabetes
or dietary cholesterol, accelerate essential fatty
acid (EFA) deficiency in rats. Further studies on
rabbits and rats have supported our earlier con-
clusions. Rabbits fed an EF A-free diet containing
1% cholesterol became severely depleted in 3
months as evidenced by loss of hair, skin lesions
and emaciation. Depleted rabbits removed from
the cholesterol diet and supplemented with 1%
corn oil showed new hair growth and improved
skin. A diet of 1% cholesterol fed to 15-day-old
rats produced a severe stress indicated by high
mortality rate. Rats fed 1% cholesterol and 1%
linoleate developed temporary scaliness of feet.
These observations support the assumption that
cholester@ transport is related to mobilization
of EFA.

1066. Purification of nucleoprotein B from
rat liver cytoplasm. Mary L. PETERMANN
AND Mary G. Hamriton.* Sloan-Kettering Inst.
for Cancer Research, New York City.
Nucleoprotein B (Cancer Research 14: 360, 1954)

accounts for most of the PNA in the microsome

fraction of rat liver. It forms a sharp ultracentrif-
ugal boundary with a sedimentation coefficient
of 45 to 78 S, depending on the viscosity of the
solution. It is extremely unstable; when the
molecule is disrupted the characteristic ultra-
centrifugal pattern is no longer seen. Previous
attempts to isolate nucleoprotein B have been
handicapped by this great instability. Recently
a stabilizing factor (SF) has been found in a

FEDERATION PROCEEDINGS

Volume 16

dialyzate of rat liver cytoplasm (J. Biophys,
Biochem. Cytol. 1: 469, 1945). SF can also be pre-
pared from calf liver. In its presence the nucleo-
protein is stable for several days in 0.25 m sucrose
and its isolation has become a practical goal,
Four major impurities must be removed: small
proteins, microsome fragments, other nucleo-
proteins and glycogen. Fractionation can be
controlled by ultracentrifugal analysis, for sta-
bility and yield, while purity is estimated best by
electrophoretic analysis. The rats are starved for
24 hr. before they are killed to reduce their liver
glycogen. The livers are perfused, forced through
a tissue press and homogenized in 6 volumes of
0.3 M sucrose in a Waring Blendor at low speed
(about 30 v.). The nuclei and mitochondria are
discarded and the microsomes disrupted by 0.5%
desoxycholate at pu 8.3. The nucleoprotein can
then be purified by repeated cycles of high and
low speed centrifugation, centrifugation into
sucrose density gradients or precipitation by 20%
ethanol at pH 6.

1067. Effect of glycocyamine and betaine on
the high energy phosphates of muscle in
multiple sclerosis. Ruta D. PETERSON AND
Joun H. Aupes.* Biochemistry Dept., Univ. of
Oregon Med. School, Portland, and Multiple
Sclerosis Clinic, Cedars of Lebanon Hosp., Los
Angeles, Calif.

It appeared possible that the extreme muscular
weakness found in patients suffering from multiple
sclerosis might be due to depletion of utilizable
energy. Administration of glycocyamine, the
immediate natural precursor of creatine in tissues,
has been shown to raise creatine phosphate levels
in skeletal muscle (Arch. Biochem. & Biophys.
54: 349, 1955). Preliminary observations indicated
better progress by patients receiving rehabilita-
tion plus glycocyamine and betaine than those
receiving only rehabilitation therapy. In a double
blind experiment, multiple sclerosis patients were
divided into 2 groups, one receiving rehabilitation
plus placebos and the other rehabilitation plus
glycocyamine and betaine. Muscle biopsies were
taken before the initiation of therapy and at
intervals thereafter. Forty-six multiple sclerosis
patients and 14 controls showed no initial differ-
ence in inorganic phosphate, creatine phosphate,
ADP + ATP, total acid soluble phosphate, total
phosphorus, creatine and dry weight. The total
nitrogen was slightly but significantly lower in
the patients with multiple sclerosis. The multiple
sclerosis patients subjected to rehabilitation plus
glycocyamine and betaine showed no significant
difference in chemical composition between the
initial muscle samples and the samples taken
(av. 7.4 mo.) after therapy. However, the patients
on rehabilitation plus placebos showed significant

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March 1956

decreases irivcreatine phosphate (P < .005) and
ADP + ATP (P < .005) (av. 6.8 mo.) after
therapy. (Supported in part by International
Minerals and Chemical Corp.)

1068. Transacetalation. CLAupE PrantTapos!,*
Cart E. ANDERSON AND E. A. Brecut.* School
of Pharmacy and Dept. of Biochemistry, Univ. of
North Carolina, Chapel Hill.

Evidence has been obtained (KLENK) that the
structure proposed for naturally occurring acetal
phosphatides (FEULGEN, BERSIN) may not be
identical with that existing in tissues. During the
chemical synthesis of acetal phosphatides for com-
parison with naturally isolated preparations, a
new method for the synthesis of 1,2-glycerol
acetals of higher fatty aldehydes was discovered.
The conventional formation of glycerol acetals by
reacting the free aldehyde with glycerol in the
presence of mineral acid yields a mixture of
acetals. Further, free aldehydes polymerize
readily. These objections were overcome by
reacting glycerol with the dimethyl acetals of the
aldehydes with catalytic amounts of sulfosalicylic
acid. A transfer of the acetal linkage to the
glycerol, accompanied by evolution of alcohol,
occurred. The alcohol was distilled off. The 1,2-
glycerol acetals were formed in high yields. The
palmital, myristal, laural, ethanal, hydroxy-
citronellal, dichloroethanal and benzal derivatives
were synthesized. Using benzilidine and ethylidine
glycerol preparations as model reactions, the
transacetalation process was investigated. The
alcohol was found to evolve in 2 stages. This
finding, coupled with the isolation of the mixed
acetals after evolution of half the alcohol, indi-
cates the reaction is a 2-stage process: Glycerol +
Dimethyl Acetal — Mixed Acetal — 1,2-Glycerol
Acetal. The first stage is acid catalyzed and prob-
ably involves a carbonium ion mechanism. The
second step is carried out with heat alone and
appears to involve an internal nucleophilic attack
leading to ring closure. The 1,2-glycerol acetal
may be converted to an equilibrium mixture of
the 1,2- and 1,3-isomers by heating in the presence
of acid. (Aided by a grant from the Life Insurance
Med. Research Fund.)

1069. Preparation of high potency concen-
trates of thyrotropic hormone. JouNn G.
PIERCE AND JosEPH F’. Nyc. Dept. of Physiologi-
cal Chemistry, Univ. of California Med. Ctr.,
Los Angeles.

Recently it has been shown that bovine thyro-
tropin can be adsorbed on the carboxylic cation
exchange resin IRC-50 and then eluted with 1
M NaCl (Herpeman. Endocrinology 53: 640, 1953;
CrIGLER AND WaueH. J. Am. Chem. Soc. 77:
4407, 1955). These adsorptions were done es-
sentially in the absence of cations and with resin

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

327

equilibrated at pu 8.0 or equilibrated with 0.01 m
sodium phosphate at pH 7.6. Crigler and Waugh
reported a potency of 1.0 to 2.0 U.S.P. u/mg. In
this laboratory it has been found that adsorption
occurs also at higher cation concentrations (Na**,
0.26-0.28 m) in the lower effective pH range of
the resin, i.e. pH 5.9 but not at px 6.4. By first
passing thyrotropin concentrates through a
column at pH 6.4 and then adsorbing the active
material on a second column at px 5.9 followed
by elution with an increasing cation concentration
gradient, one obtains material of approx. 5 U.S.P.
u/mg. The resin was finely ground IRC-50. The
activity was measured by P** uptake in chick
thyroids. Starting material for the chromato-
graphic experiments was prepared by the initial
extraction and acetone precipitations of Ciereszko
(J. Biol. Chem. 160: 585, 1945). Chromatography
of such starting material allows recovery of
reasonable amounts of 5 u/mg material without
use of excessively large columns. (Supported in
part by grants from the Public Health Service
and the American Medical Association.)

1070. Sources and electrophoretic properties
of normal synovial fluids. Warp PiaMaAn,
Davin Piarr,* Francis Patton* anp Howarp
Houiey.* Univ. of Alabama Med. Ctr.,
Birmingham.

Synovial fluids from 14 normal individuals
differ in a number of electrophoretic properties
from those of rheumatoid arthritics and from
serums. Normal fluids frequently contain 2 com-
ponents moving faster than albumin. The mobility
(relative to albumin) of the faster moving com-
ponents, and particularly of the alpha-two and
beta-globulins, differ significantly from those of
arthritic joint fluids. Since normal fluids can be
obtained at best in only small amounts (0.2-0.5
cc), other sources of synovial fluid have been
investigated. Examination of post-mortem fluids
obtained at autopsy was made. The properties of
these fluids resembled closely those taken from
normal individuals, except for a greater variability
of the dialyzable NPN. Eight of the 11 fluids had
2 components moving faster than albumin. With
proper discrimination, post-mortem synovial
fluids may be substituted for normal fluids in
investigative studies. Synovial fluids collected
from 12 cattle were studied in similar fashion.
Three of the fluids exhibited 2 fast moving com-
ponents. Studies are in progress on the nature
and significance of the fast moving components.
(Aided by the Public Health Service, the Arthritis
and Rheumatism Fndn. and the John R. Irby
Fund.)

1071. a-Keto acid keto-enol tautomerase.
Burnetr M. Pirr* anp W. Eveene Knox.
Cancer Research Inst., New England Deaconess

328

Hosp., and Dept. of Biological Chemistry, Harvard

Med. School, Boston, Mass.

An enzyme which catalyzes the keto-enol tau-
tomerization of 8-arylpyruvic acids is found in
certain animal tissues. It has been purified 40-fold
from hog liver and kidney by ammonium sulfate
and alcohol fractionation, and separated from
tyrosine transaminase and p-hydroxyphenyl-
pyruvate oxidase. The rates of tautomerization
are measured spectrophotometrically, trapping
the enol tautomer with borate to form a strongly
absorbing borate complex. The reaction can be
followed in either direction using equilibrated
aqueous (keto) or freshly dissolved (enol) solu-
tions of the substrate. The position of equilibrium
is unaffected by the enzyme. The reaction follows
reversible first order kinetics, shows a linear
rate dependence on enzyme concentration, and a
broad rate maximum around pH 7. The enzyme-
catalyzed reaction is accelerated by phosphate as
compared with acetate and is somewhat inhibited
by arsenate. The spontaneous reaction is more
accelerated by arsenate than by phosphate.
Phenyl and p-hydroxyphenyl pyruvates are
rapidly tautomerized by the enzyme, m-hydroxy-
phenyl and p-methoxyphenyl pyruvates less
rapidly, and indole and imidazole pyruvates
slowly or not at all. No evidence of tautomerase
activity with aliphatic a-keto acids has thus far
been obtained. There are no indications of a metal
function in the tautomerase, but the reversibility
of its mercury inhibition by cysteine indicates
that a thiol group is required for activity. The
enol-borate complexes may be used for convenient
assays of B-aryl-a-keto acids and with the enzyme
may be used for the separate determination of the
keto and enol forms of these compounds as biologi-
cal intermediates. (Supported by Atomic Energy
Commission Contract no. AT(30-1)901 with the
New England Deaconess Hosp.)

1072. PhSsphorylation of mitochrome coupled
with electron transport over cytochrome c.
C. Davin Potts anp H. W. SHMUKLER (intro-
duced by Davin L. Drasxtin). Aviation Med.
Acceleration Lab., U. S. Navy, Johnsville, and
Dept. of Biochemistry, Grad. School of Medicine,
Univ. of Pennsylvania, Philadelphia.
Following the demonstration of the acceleration

effect of mitochrome on the rates of DPNH oxida-

tion and phosphate esterification in a system
with cytochrome c and intact mitochondria (PoLis

AND SHMUKLER. Abstracts, Division of Biol. Chem.,

Am. Chem. Soc., 128th meeting, 19 C, 1955), the

experimental work was extended to reproduce the

reactions in solutions free of mitochondria. Al-
though purified preparations of mitochrome and
cytochrome c were inactive with DPNH, less pure
mitochrome preparations with a trace component

FEDERATION PROCEEDINGS

Volume 15

showing a band at 556 my (reduced state) reacted
rapidly to reduce cytochrome c and oxidize
DPNH. After formation of DPN, the reduced
cytochrome c was slowly reoxidized in this system,
In the presence of P*, the mitochrome periodically
removed from the reaction mixture by isoelectric
precipitation at pu 5.1 showed a gradual uptake
of P*? to a maximum value of 1 atom of P/M of
mitochrome. In the absence of cytochrome c or
electron transport there was no significant uptake
of P* by the chromoprotein. With the addition of
ADP and an enzyme factor in fresh preparations
of myokinase to the above system the data ob-
tained suggest a transfer of P* from the mito-
chrome to an organically bound fraction. A further
implication of mitochrome in aerobic phosphoryla-
tion was indicated by the inhibition of the electron
transfer mechanism of the mitochrome-cyto-
chrome c with Pentothal, supported by the spec-
trophotometric findings.

1073. Respiratory activity of normal and
bruised red tart cherry (Prunus cerasus).
Rosert L. Potuack AND CLAupE H. HI.is
(introduced by Sam R. Hoover). Eastern Utili-
zation Research Branch, Agric. Research Service,
U.S. Dept. of Agriculture, Philadelphia, Pa.
Previous studies at this laboratory have shown

that various biochemical changes take place when

harvested red tart cherries are bruised. At the
present, however, there is little fundamental
information available concerning how these
changes take place or how they are related to the
physiology of the normal living tissue. Respiration
studies were conducted on normal and bruised
cherries using the Warburg apparatus with vessels
large enough to accommodate several cherries.
The determinations were performed, routinely,
on 2 cherries per vessel. Each set acted as its own
control in that, after having determined the
oxygen and carbon dioxide exchanges of the
normal fruit, the cherries were removed from their
respective flasks, bruised, returned to the same
flasks and the respiratory exchanges again deter-
mined, Experiments continued for periods up to
6 hr. on normal samples showed that the respira-
tory activities were linear. The respiratory
quotient rose with increasing maturity of the
normal fruit and reached the value of 1.95 for the
most mature sample. Following bruising the
increase in carbon dioxide output greatly exceeded
the increase in oxygen utilization. In terms of
ul/gm fresh tissue/hr. the oxygen consumption
increased approximately 50% following bruising,
whereas the CO output increased approximately

126%. The respiratory quotient rose from an

average of 1.80 for the unbruised fruit to 2.47

after bruising.

tri
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ume 15

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March 1956

1074. Formaldehyde fixation by Propionibac-
terium arabinosum as determined by mass
analysis of C'3-propionate. Srymour H.
PoMERANTz (introduced by W. F. H. M. Mom-
MAERTS). Dept. of Biochemistry, Western Reserve
Univ. Med. School, Cleveland, Ohio.

Leaver and Wood (J. Cell. Comp. Physiol. 41:
Suppl. 1, 225, 1953) found that P. arabinosum
incorporates C'4-formaldehyde into every position
of all isolated products (glucose or glycerol sub-
strate). The question arose whether the propionate
formed was a mixture of different types of singly
labeled molecules, or of singly and doubly labeled
molecules or contained triply labeled molecules.
The last would imply a total synthesis from
formaldehyde carbon. With Wood’s method of
mass analysis (J. Biol. Chem. 194: 905, 1952) one
can differentiate the types of propionate on the
basis of their differences in mass. C!-formalde-
hyde has been used and the propionate converted
to propylene gas. From the spectrum obtained in
the mass spectrometer we determined the amounts
of unlabeled (mass 42), singly (43), doubly (44),
and triply labeled (45) propylene. The calculations
involved were shown to be valid by mass analysis
of propylene derived from artificial mixtures of
severally labeled propionates. With a glucose
substrate it was found that 5.4% of the propionate
was singly labeled; the amount of doubly and
triply labeled material was <0.1%. On chemical
degradation, 26% of the C!* was in the carboxyl
group with the remainder equally divided between
the other carbons. This indicates that the
propionate is a mixture of 3 singly labeled types
and unlabeled propionate. Analogous results were
obtained with the acetate formed in the
fermentation.

1075. Amino acid-nucleotide complexes in
Schmidt-Thannhauser digests of ribo-
nucleic acid. JosEPpH L. PoTrER* AND ALEX-
ANDER L. Dounce. Dept. of Biochemistry, Univ.
of Rochester School of Medicine and Dentistry,
Rochester, N. Y.

The alkaline hydrolysis of mammalian ribo-
nucleic acids (RNA) prepared by the method of
Kay and Dounce or of yeast RNA prepared by the
method of Johnson and Harkins does not lead to
the complete recovery of nucleotide material as
mononucleotides by using the technique of ion-
exchange chromatography. In addition to the
mononucleotides, alkali-resistant material may
be eluted from the columns by using stronger acid
than is required for the removal of the mono-
nucleotides. This additional material generally
appears as 3 distinct peaks following the mono-
nucleotide peaks. The material corresponding to
each peak has been found to contain nucleotides,
predominantly of purine type, and either amino

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

329

acids or short peptides bound to them. The ratio
of amino acid to nucleotide is apparently some-
what less than one. The material corresponding
to each peak is ninhydrin-negative until hy-
drolyzed by heating with acid. The possibility
that the new fractions might be contaminated
with free amino acids or protein seems to be
excluded by the fact that the latter substances
are not bound by the column under the conditions
of elution of the mononucleotides. In view of the
preceding material and the fact that the original
RNA can be obtained biuret-negative, the hy-
pothesis is presented that the amino acids are
bound to phosphate groups of the nucleotides in
phosphoamide-type linkage. It appears possible
that the bound amino acids represent intermediate
stages in peptide-chain synthesis.

1076. Phosphorylation of adenine nucleotides
by mitochondria. BERTON C. PRESSMAN
(introduced by H. A. Larpy). Inst. for Enzyme
Research, University of Wisconsin, Madison.
When rat liver mitochondria phosphorylate oxi-

datively with adenine nucleotides as the only

added phosphate acceptor, a sudden drop in the
rates of both oxidation and phosphorylation
occurs at some critical period following 43-6 min.
after the addition of the adenine nucleotide. This
effect has been studied both by the conventional
manometric techniques and following substrate
and inorganic phosphate concentrations by with-
drawing intermittent samples from open vessels.

The phenomenon depends on the amount of mito-

chondria, the equivalent of at least 0.2 gm

liver/ml. incubation mixture being required, and

is rendered more acute by the presence of 0.002 m

versene. The basic effect is obtained with a-keto-

glutarate, §-hydroxybutyrate, t-malate or L-

glutamate as substrate, adenosine mono- or

or diphosphate as phosphate acceptor, in the

presence or absence of 0.01 m fluoride or 0.02 m

malonate and is not markedly influenced by phos-

phate, adenosine nucleotide or magnesium con-
centrations. It is reversed by addition of hexo-
kinase and glucose, dinitrophenol, desoxycholate,
mitochondrial acetone powder extracts, or puri-
fied myokinase. If mitochondria which have
entered the phase of inhibited respiration were
replaced in the reaction mixture by fresh mito-
chondria, respiration of the latter was unimpaired
indicating that nothing inhibitory to the mito-
chondria had accumulated in the reaction mixture.

1077. Separation of sulfhydryl derivatives on
paper. C. A. Price (introduced by Bernarp S.
SrrREHLER). Dept. of Horticulture, Purdue Univ.,
Lafayette, Ind.

A method has been developed for the chromato-
graphic separation of the N(4-hydroxy-l-

330

naphthyl)-isomaleimide (HNI) derivatives of
naturally occurring soluble sulfhydryl compounds
by the combination of the methods of Gutcho and
Laufer (in: Glutathione, New York: Acad. Press,
1954) and Tsou, Barrnett and Seligman (J. Am
Chem. Soc. 77: 4613, 1955). A small sample of tissue,
typically 100 mg wet weight, is ground in an
excess of mm HNI in 50% ethanol. After reaction
and centrifugation 10-100 ul of the supernatant
were spotted on Whatman no. 20 paper and the
chromatogram developed with 95% ethanol.
The HNI-sulfhydryl compounds were then de-
tected by spraying with tetra-azotized diortho-
anisidine. The limit of visual sensitivity is lower
than 10 mum while 100 mym give conveniently
bright and stable blue or red spots on a yellow
background. With plant samples, including pea
seedlings, spinach leaves and _ various in-
florescences, up to 8 spots were observed including
the derivatives of glutathione, cysteine, and
possibly coenzyme A. A survey of the range and
relative concentrations of sulfhydryl compounds
within a selected group of plants has been con-
ducted. (Dr. Arnold M. Seligman kindly provided
a sample of HNI.)

1078. Enzyme induction and _ tryptophan
metabolism in isolated perfused rabbit
liver. Joun B. Price* anp L. S. Drerricu.
Depts. of Surgery and Biochemistry, College of
Physicians and Surgeons, Columbia Univ., New
York City.

Incorporation of radioactive amino acids into
hepatic and plasma proteins readily occurs in the
isolated, perfused liver (Miller et al. J. Exper.
Med. 94: 431, 1951; Prudden et al. J. Lab. & Clin.
Med. March, 1956). Enzyme induction employing
the above system has not been previously demon-
strated. Using the technique of Young, Prudden
and Stirman (J. Lab. & Clin. Med. 46: 155, 1955)
rabbit livers were perfused with a mixture of
blood, afhino acids, glucose and L-tryptophan for
6-8 hr. The activity of the tryptophan peroxidase-
oxidase system (I) was follows by: 1) assay of
enzymatic activity in the liver before and after
perfusion, 2) rate of appearance of aromatic
amines in the perfusate, 3) chromatographic
analyses of the perfusate. Two to 4-fold increases
in I were routinely found. pi-ethionine reduced
this response when given during the perfusion or
to the animal prior to perfusion. This inhibition
can be reversed by t-methionine. Paper chroma-
tography of the deproteinized, concentrated
perfusate (plasma) revealed a large number of
tryptophan metabolites. Kynurenine and
tryptophan with their n-alpha acetyl derivatives
together with kynurenic acid and anthranilic acid
derivatives were major components. Neither 3-
hydroxykynurenine compounds nor xanthurenic

FEDERATION PROCEEDINGS

Volume 1§

acid were found. Several hydroxylated indoles
were present, the majority of which have the UV
spectra and chemical color reactions of §
hydroxyindoles.

1079. Quantitative studies on the metabolism
of tryptophan in patients receiving
isoniazid therapy. J. M. Price, R. R. Brown,
AND FRANK C. Larson (introduced by J. A,
MILLER). Cancer Research Hosp. and VA Hosp.,
Univ. of Wisconsin Med. School, Madison.
Patients with minimal tuberculosis were given

a test dose of L-tryptophan at weekly intervals

and the urines for the day before and the day after

the amino acid supplementation were analyzed
quantitatively for several tryptophan metabolites,

After the first test dose the subjects were started

on isonicotinic acid hydrazide therapy, increasing

the dose from 300 to 750 mg daily in a period of

4 wk. The patients responded to the tryptophan

supplements like normal, nontuberculous subjects

prior to taking the drug. In 3-5 wk. of therapy
there was a progressive increase in the urinary
excretion of kynurenine and acetylkynurenine in
response to the 2-gm dose of tryptophan, with
values reaching over 20 times pretreatment levels,
There was a much smaller, progressive increase
in xanthurenic excretion, while the yields of
kynurenie acid, N-methyl-2-pyridone-5-carbox-
amide, o-aminohippuric acid, and anthranilic acid
glucuronide were somewhat below normal at all
times. Paper chromatography indicated high
urinary levels of 3-hydroxykynurenine and its

N-a-acetyl derivative when kynurenine excretion

was increased. No other unusual spots were noted.

Supplemental thiamine, riboflavin and nicotinic

acid had little effect on the results, but adminis-

tration of pyridoxine hydrochloride resulted in a

prompt restoration of normal tryptophan metabo-

lism. The results indicate that, at the dosage used,
isoniazid strongly inhibited the metabolism of
kynurenine and 3-hydroxykynurenine through

reactions involving kynureninase and _trans-
aminase, both of which require pyridoxal
phosphate.

1080. Labeling of different portions of phos-
phoglyceride molecule in rat liver and
brain slices. E. T. PrircHarp (introduced by
F. C. Heacy). Dept. of Biochemistry, Univ. of
Western Ontario, London, Canada.

When slices of rat liver or brain are allowed to
respire in a Krebs-Ringer bicarbonate medium
containing suitable radioactive precursors, differ-
ent portions of the phosphoglyceride molecules
present in the acid-insoluble lipid may be labeled
independently. For liver slices, the activity of the
total acetone-insoluble lipid was found to be in
the order: acetate-1-C'4 > glycine-2-C™ > glye-

Marc

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1081.
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FROWN,
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March 1956

erol-1-C'*, whereas for brain slices the order
was: glycine-2-C“ > acetate-1-C'* > glycerol-
1-C'*. Hydrolysis experiments showed that, for
both liver and brain slices, acetate-1-C' labeled
the fatty acid portion of the phosphatide molecule
and not the nonfatty acid portion, whereas gly-
cine-2-C and glycerol-1-C™ labeled the nonfatty
acid portion of the molecule, and the fatty acid
portion only to an insignificant degree. Using the
hydrolysis and chromatographic procedure of
Dawson (Biochim. et Biophys. Acta 14: 374, 1954),
together with degradation of the hydrolysis
products and subsequent rechromatography, it
was found that glycine-2-C" labeled the nitrogen-
containing portion of the phosphoglyceride, and
not the glycerol portion. In both liver and brain
slices the activity was in the order: phosphatidyl
serine > phosphatidyl ethanolamine > phos-
phatidyl choline. Glycerol-1-C' labeled the
glycerol moiety of the phosphoglyceride molecule
and not the base. For liver slices the activity was
in the order: phosphatidyl choline > phosphatidyl
serine > phosphatidyl ethanolamine, and for
brain slices the order was: phosphatidyl
serine > phosphatidyl choline > phosphatidyl
ethanolamine.

1081. Spectral changes occurring during
hydrolysis of di-deoxyribonucleotides by
phosphodiesterase. M. Privat pE GARILHE*
and M. Laskowski. Dept. of Biochemistry,
Marquette Univ. School of Medicine, Milwau-
kee, Wis.

It has been a controversial issue (BEAVEN
etal. In: CHARGAFF AND Davipson. Nucleic Acids,
vol. I, p. 493) whether the optical density of di-
and trinucleotides represents the sum of the
optical densities of the component mononucleo-
tides. The availability of purified phospho-
diesterase (Biochim. et Biophys. Acta 18: 370, 1955)
made it possible to measure directly the optical
effect resulting from hydrolysis of an internucleo-
tide linkage. The solution of a dinucleotide in
0.1 m glycine-NaOH buffer, po 9, containing
0.03 m MgSO, was placed in a 1 em wide Beckman
cell at 37°. The increase in optical density after
addition of enzyme was recorded at 1 min. inter-
vals. Zero order kinetics persisted until about
50% of the substrate was hydrolyzed. When the
method was applied to a mixture of oligonucleo-
tides, the final increase in optical density was
about 12% of the original optical density. The
magnitude of the effect depends on the nature of
the dinucleotide. On the whole, the increase in
optical density is small with pyrimidine dinucleo-
tides and large with those containing a purine. The
Maximum difference in spectrum between the
original and hydrolyzed compounds did not
necessarily occur at a wavelength corresponding
to the maximum of the original compound. Optical

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

331

changes of the same order of magnitude have been
observed on hydrolysis of dinucleotides (pXpY)
and dinucleoside monophosphates (XpY); the
rate of hydrolysis, however, was much slower for
the latter. This result was confirmed by
chromatography. (Supported ‘by the Atomic
Energy Commission.)

1082. A new acid-stable component of DNA
derived from formate-C'%, Wiiiiam H.
Prusorr AND LaszLto G. LasTHa (introduced
by WERNER BERGMANN). Dept. of Pharmacology,
Yale Univ., New Haven, Conn.

Formate-C'* was incorporated preferentially
in vitro into the thymine portion of DNA of rabbit
bone marrow and Ehrlich ascites tumor cells of
mice. The DNA derived from the tumor cells was
digested with perchloric acid (12 nN) at 100° for
1 hr. and chromatographed in an isopropanol-HCl
system. The specific activity of DNA-thymine was
increased markedly by the addition of uracil
deoxyriboside and to a much greater extent by
cytosine deoxyriboside (Proc. Am. Chem. Soc.,
128th meeting, Sept. 1955, p. 6C). Uridine and
cytidine were as effective as the corresponding
deoxynucleosides, presumably because of a rapid
conversion of the riboside to the deoxyriboside
(cf. I. A. Rose anv B. 8. Scuwercert. J. Biol.
Chem. 202: 635, 1953). The cytosine area on the
chromatogram of the perchloric acid digest of the
DNA was significantly radioactive; however, the
radioactivity could be separated from the non-
radioactive cytosine in a butanol-ammonia system
(Rf = 0.0-0.1). When re-chromatographed in the
isopropanol-HCl system, the mobility of the
radioactive component again corresponded to that
of cytosine. The addition of uridine or cytidine
to the incubation mixtures (tumor cells, buffer
and formate-C") resulted in a marked increase
in the radioactivity of the material; the increment
was of the same magnitude as that observed in
the thymine of the DNA. On the basis of paper
electrophoresis this radioactive material was not
5-methyleytosine, 5-hydroxymethylcytosine, 5-
carboxycytosine, orotic acid or uracil. The data
suggest the existence of a new acid-stable com-
pound which contains a carbon atom that can be
derived from formate. This compound is either a
component of DNA or is associated closely with it.

1083. Biosynthesis of abnormal proteins in
multiple myeloma. FRANK W. PutNaM, FRANZ
Meyer* anp Arxo Mryake.* Dept. of Bio-
chemistry, and Argonne Cancer Research Hosp.,
Univ. of Chicago, Chicago, Ill.

To ascertain if Bence-Jones protein (BJ) is an
intermediate in serum globulin synthesis, a patient
excreting BJ and having a myeloma globulin of the
‘y’? type was injected with 400 ue of pi-glutamic
acid-1-C'*. Blood and urine samples were taken

332

frequently by indwelling catheters. The BJ and
y proteins were isolated, characterized physico-
chemically and their radioactivity determined.
The radioactivity was also determined of: 1)
expiratory CO:, 2) urinary urea CO: (urease),
8) urinary amino acid CO: (ninhydrin vs. specific
decarboxylase) and 4) serum tL-glutamic acid
(decarboxylase). (1) was maximal at 25 min.,
declining with a & of 85 min. and accounting for
3.1% of the injected activity; (2) and (3) were
maximal at 2.5 hr., but (3) declined more rapidly
(like 1) and 99.9% of the activity of (3) was due to
the p-isomer. A lag of 40 min. occurs before labeled
protein is found in serum (7) or urine (BJ) during
which (4) declines abruptly. The activity of BJ
was maximal at 5.5 hr. and then declined sharply
whereas the activity of y was maximal at 10 hr.
and fell off gradually. The highest activity of BJ
was 10 times that of y. The data indicate that
L-glutamic acid is rapidly metabolized and the
p-isomer is rapidly excreted. Although previous
work with stable isotopes (J. Biol. Chem. 212:
361, 371, 1955) suggested the synthesis of BJ
and y might be independent, the present more
accurate kinetic data are compatible with the
hypothesis that BJ is a precursor or abortive
product of serum globulin synthesis.

1084. Hyperheparinemia observed clinically
and after intravenous injection of papain
into rabbits. ARMAND J. Quick. Dept. of Bio-
chemistry, Marquette Univ., Milwaukee, Wis.
Heparin as a cause of hemorrhage is rare which

makes the following case significant. A woman,
aged 22, with a history of mild bleeding since
childhood required 34 transfusions after the
birth of her 1st baby and 28 after her 2nd child. A
study of her blood 2 months after the 2nd parturi-
tion showed clotting time ~, prothrombin time
14 sec., platelet count 210,000, thrombin time 43
sec. (normal, 3 sec.) and after adding protamine
sulfate, 7 sec. Six weeks later the findings were
essentially unchanged but oddly no signs of a
hemorrhagic tendency were manifested. The find-
ings suggest that the coagulation defect is caused
specifically by a marked hyperheparinemia. An
outpouring of heparin into the blood is known to
occur in peptone and anaphylactic shock. It was
found that it is also caused by injecting papain
intravenously into rabbits. Heparinemia, which
was demonstrable 5 min. after the injection,
became progressively more marked. Bleeding
especially from venipunctures became profuse but
was promptly controlled by the injection of pro-
tamine sulfate which reduced the heparin titer of
the blood. No significant change in the platelet
count or the concentration of fibrinogen was
noted after injecting papain. (Aided by a grant
from Wisconsin Heart Assoc.)

FEDERATION PROCEEDINGS

Volume 16

1085. Inhibition analysis of amino acid in.
corporation into protein of the Ehrlich
ascites cell. M. Rasinovitz, M. E. Ouson*
AND D. M. GreEenBerG. Dept. of Physiological
Chemistry, Univ. of California School of Medicine,
Berkeley.

Three types of amino acid analogues have been
found which interfere with incorporation of radio-
active amino acids into protein; 1) those which
interfere only with the incorporation of their cor-
responding metabolite, and are quantitatively in-
corporated in their place, e.g. ethionine; 2) those
which block the incorporation of other amino acids
as well as their specific metabolite, e.g. O-methyl-
threonine. Inhibition by this antagonist is relieved
only by its metabolite, isoleucine; 3) those which
inhibit the formation by the cell of an amino acid
essential for protein synthesis. Methionine sulf-
oximine appears to belong to the last category, for
its inhibition of incorporation of a variety of amino
acids is relieved by glutamine in a noncompetitive
manner. It does not interfere with the incorpora-
tion of radioactive glutamine into protein. Both
O-methylthreonine and methionine sulfoximine
only partially inhibited the incorporation of radio-
active phenylalanine and leucine. O-methylthre-
onine did not inhibit that portion of incorporation
which was resistant to inhibition by methionine
sulfoximine. These results imply that incor-
poration into the uninhibitable fraction may be
due to some process other than protein synthesis.
In addition to preventing inhibition by methi-
onine sulfoximine, glutamine could relieve it
when subsequently added to the inhibited system.
Moreover, after preincubation of the cells with
methionine sulfoximine, the addition of glutamine
stimulated amino acid incorporation to a level
20-50% above that observed after the cells had
been preincubated in buffer alone. This over-
compensation may be due to the accumulation of
some intermediate whose concentration usually
limits the rate of protein synthesis. (Aided by
grants from the Damon Runyon Memorial Fund
for Cancer Research, Inc. and the Cancer Re-
search Funds of the Univ. of California.)

1086. Metabolism of formiminoglycine and
glycine by Clostridium acidi-urici. JEssE C.
RaBINOWITZ AND W. E. Pricer, Jr.* Natl.
Insts. of Health, PHS, Bethesda, Md.
Formiminoglycine (NH=CH—NH—CH:—

COOH, FIG) has been identified as the final prod-

uct of the degradation of xanthine and 4-amino-

imidazole by extracts of lyophilized cells of

Clostridium cylindrosporum and Clostridium acidi-

urict (RABINOWITZ AND Pricer. Federation Proc.

14: 266, 1955). With washed cells of C. acidi-urici

this product is further metabolized according to

the equation: FIG — CO. + CH;COOH + 2NH;.

ume 15

id in.
hrlich
)LSon*
logical
dicine,

e been
“radio-
which
‘ir cor-
ely in-
) thoge
oO acids
1ethyl-
slieved
which
10 acid
e sulf-
ry, for
amino
etitive
rpora-
. Both
ximine
radio-
ylthre-
ration
‘ionine
incor-
ay be
thesis,
methi-
ave it
ystem.
3 with
amine
, level
Is had
over-
‘ion of
sually
ed by
Fund
r Re-

. and
sSE C.
Natl.

CH:—
prod-
mino-
lis of
acidi-
Proc.
uric
ing to
2NH;.

March 1956

Glycine alone is not fermented by the washed
cells; however, it is fermented to yield acetic acid
and carbon dioxide when FIG is added. FIG ap-
pears to act catalytically since from 1.4 to 3.7 Mm
of glycine are utilized per mole of FIG consumed.
Formiminoglutamic acid and formiminoaspartic
acid are not fermented, nor can they replace FIG
in stimulating glycine utilization. Formylglycine,
formate, formaldehyde and formamide are also in-
active in stimulating glycine utilization. Using
synthetic samples of FIG-C", it was found that
the carboxyl carbon of acetic acid is derived from
the methylene carbon of FIG and that both the
methyl carbon of acetic acid and carbon dioxide
are derived from the carboxyl and formimino
carbons of FIG. When mixtures of glycine-C* and
FIG were fermented, it was found that the methyl-
ene carbon of glycine is converted to the carboxyl
carbon of acetic acid, while the carboxyl carbon
of glycine appears as carbon dioxide and the
methyl carbon of acetic acid. Extracts of the
organism have been obtained which form acetic
acid from FIG when supplemented with Fe**,
sulfide and a boiled cell extract.

1087. Methyl synthesis in the rat from for-
mate intramolecularly labeled with C“ and
deuterium. JuLtIAN R. RacHELE anp HvuGo
AxrsI.* Dept. of Biochemistry, Cornell Univ. Med.
College, New York City.

In a previous study of labile methyl synthesis in
the rat with formate, labeled intermolecularly
with C“ and with deuterium at a level of 73 atom %
(REssLER, RACHELE AND DU VIGNEAUD. J. Biol.
Chem. 197: 1, 1952), the results were taken to indi-
cate that the carbon of formate is utilized for
choline methyl synthesis without loss of its hydro-
gen. In a later, unpublished experiment, when
formate, similarly labeled intermolecularly with
C“ and with deuterium, but with 97 atom %
deuterium, was given to rats, the incorporation of
deuterium into choline and creatine methyl groups
was much greater in relation to the C'‘ than in the
first experiment. These results were attributed to
hydrogen isotope selection in the over-all metabo-
lism of formate, and this belief was supported by
the finding that the deuterium content of urinary
formate was considerably enriched in relation to
C™ as compared to the ratio of these isotopes in
the administered compound. In the present study,
formate was doubly labeled intramolecularly with
C and deuterium so that the C atoms were
bonded to deuterium atoms. The virtual elimina-
tion of hydrogen isotope fractionation with this
type of multiple labeling is indicated by the result
that the urinary formate has the same D/C" as the
administered formate. The D/C" in the methyl
groups of choline and creatine support the earlier

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

333

contention that methyl synthesis from formate
occurs with neither loss nor labilization of the
hydrogen of formate.

1088. Enzymatic and nonenzymatic conver-
sion of hydroxyproline to pyrrole-a-car-
boxylic acid. A. N. RADHAKRISHNAN* AND
Auton Merster. Natl. Cancer Inst., Natl. Insts.
of Health, PHS, Bethesda, Md.

Purified sheep kidney p-amino acid oxidase
preparations in the presence of catalase oxidize
allohydroxy-p-proline and hydroxy-p-proline
(consuming 0.5 mM of oxygen) to pyrrole-a-car-
boxylic acid: (PCA). Enzymatically formed PCA
was isolated in analytically pure form in good
yield; m.p., chromatographic behavior, infrared
and ultraviolet spectra of the isolated material
and a synthetic sample of PCA were identical.
Both samples of PCA were catalytically reduced to
products, identified by paper chromatography in 5
solvents as proline and a-aminovaleric acid (AV);
under these conditions, proline also gave AV;
hydroxyproline gave proline and AV. PCA ex-
hibits high characteristic ultraviolet absorption
with a band at 266 my in 0.1 Nn HCl. When the en-
zymatic oxidation was followed spectrophoto-
metrically very little PCA was formed. However,
on acidification, an approximately stoichiometric
quantity of PCA was formed. The evidence sug-
gests formation of an intermediate, presumably
A’-pyrroline-4-hydroxy-2-carboxylic acid. The
intermediate, which has been visualized by paper
chromatography, is slowly converted to PCA at
pH 8.5; in acid conversion is much more rapid.
When oxidation is carried out with oxidase prepa-
rations low in catalase activity, only traces of the
intermediate and PCA were detected. This re-
action, in which 1 m of oxygen is utilized and 1 m
of CO: is evolved, is being investigated. We have
also observed that the nonenzymatic oxidation of
hydroxyproline by hydrogen peroxide in the pres-
ence of copper sulfate and alkali results in forma-
tion of PCA, which appears to represent the major
oxidation product.

1089. Galactolipid metabolism. Norman §&.
Rapin, F. Barsara Martin, JAMES R. Brown
AND JOHN R. ALLEN (introduced by Morais A.
Lipton). Kadioisotope Service, VA Research
Hosp., and Dept. of Biochemistry, Northwestern
Univ. Med. School, Chicago, Til.

C'*-galactose was injected into young rats and
the activity in the galactose of brain lipid fractions
was determined as a function of time. ‘Strandin’
activity was determined by dialyzing the aqueous
extract of the lipid solution, hydrolyzing the non-
dialyzing material and oxidizing to mucic acid,
which was then counted. Phospholipids were re-
moved with Florisil and ionic lipids by mixed ion-

334

exchange resins. ‘Cerebroside’ activity was de-
rived from the nonionic lipid effluent as above.
‘Cerebroside sulfate’ activity was derived by
eluting the resins with LiOAc—CHCl;—EtOH—
H.O, concentrating and precipitating the Ba salt.
The ‘strandin’ and ‘cerebroside’ curves reached
their peaks rapidly and declined to half the ac-
tivity in roughly 10 days, showing that breakdown
occurs simultaneously with synthesis. The ‘cere-
broside sulfate’ curve rose more slowly and leveled
off, indicating lack of breakdown. A similar curve
was obtained with S*O,. C'-mucic acid was ob-
tained from a lipid dissociated by CHCl;—
MeOH—HCI from residual extracted brain. Con-
siderable activity was also found in nongalactose
fractions. Distribution studies with C'‘-lignoceric
acid were carried out. (Supported by a grant from
the Multiple Sclerosis Fndn. of America, Chicago,
Dr. Lewis Pollock, responsible investigator.)

1090. Anomalous behavior of muscle and yeast
3-phosphoglyceraldehyde dehydrogenase
toward _ several sulfhydryl reagents.
GaLeE W. Rarter (introduced by Rocrer M.
Herriotr). McCollum-Pratt Inst. and Dept. of
Biochemistry, School of Hygiene and Public
Health, Johns Hopkins Univ., Baltimore, Md.
The sulfhydryl content of muscle and yeast

3-phosphoglyceraldehyde dehydrogenase has been
estimated with the following reagents: p-chloro-
mercuribenzoate, 2,6-dichlorophenolindophenol,
and o-iodosobenzoate. Ten moles of p-chloro-
mercuribenzoate was found to react per m of
muscle protein (in general agreement with a
previous estimate using this reagent, SEGAL AND
Boyer, J. Biol. Chem. 204: 265, and with the half-
cystine content of the protein of 10.7 residues per
118,000 gm protein, VELICK AND Ronzont, J. Biol.
Chem. 173: 627). Only 2 m of the reagent reacted
per M of the yeast protein. The dye, 2,6 dichloro-
phenolindophenol, and o0-iodosobenzoate both re-
acted toythe extent of about 10 M/m of muscle
protein. This is about twice the reducing equiva-
lents expected from the half-cystine content of the
protein. Evidence will be presented that the re-
action of dye and protein is not an oxidation of the
protein sulfhydryls, but rather a chemical com-
bination of dye and protein. This dye-protein
complex shows a diaphorase-like activity toward
reduced DPN. About 2 and 6 Mo, respectively, of
dye and o-iodosobenzoate reacted per Mm of the
yeast protein. The o-iodosobenzoate-treated
muscle protein no longer catalyzes the oxidation
of glyceraldehyde, but it does show phosphatase
activity for acetyl phosphate. The ability of the
treated protein to oxidize glyceraldehyde is re-
stored after reaction with cysteine.

1091. Effect of epinephrine and glucagon on
the reactivation of phosphorylase in liver

FEDERATION PROCEEDINGS

Volume 16

homogenates. THEODORE RA.u,* Ear. W,

SUTHERLAND AND JACQUES BERTHET.* Dept. of

Pharmacology, Western Reserve Univ., Cleveland,

Ohio.

The concentration of active phosphorylase in
liver represents a balance between inactivation
(dephosphorylation) by inactivating enzyme
(phosphorylase phosphatase) and reactivation by
dephosphophosphorylase kinase. In liver slices,
epinephrine and glucagon (H-G factor) displace
this balance in favor of the active phosphorylase,
It has now been possible to demonstrate a similar
effect of epinephrine and glucagon in liver homoge-
nates. Dog (or cat) liver slices were incubated in
a salt-buffer medium at 37° for 15 min., at which
time the concentration of active phosphorylase
had reached a low value. The slices were chilled
and rinsed in sucrose and a 25% homogenate was
prepared using an all-glass homogenizer. After
brief centrifugation at low speed, aliquots of the
homogenate were incubated with buffer, magne-
sium ions and adenosine triphosphate in the pres-
ence or absence of epinephrine or glucagon. In
the presence of the hormones, the rate of phos-
phorylase reactivation was increased 2-4 times,
and 50-70% of the phosphorylase present in the ho-
mogenate was reactivated in 10 min. at 30°. When
purified inactive dog liver phosphorylase was added
to these homogenates, it was also reactivated and
the rate of reactivation was likewise stimulated by
the hormones. The stimulation of phosphorylase
reactivation was related to the concentration of the
hormones; half maximal stimulation was achieved
with l-epinephrine at approximately 1 part/50
million (1 X 10-7 M) and with glucagon at even
lower molar concentrations.

1092. Effect of autoperfusion of the liver on
detoxication of thiopental sodium. A. M.
Rappaport, G. Y. Hrrakr, B. RosenFexp, W.
G. B. Cassetman, C. R. Cowan anv J. LANG
(introduced by D. A. Scorr). Dept. of Physiology,
Univ. of Toronto, Toronto, Canada.

Experiments decreasing the hepatic circulation
in dogs and resulting in significant delay of thio-
pental detoxication are reported. A method has
been worked out for arterial or venous autoper-
fusion of the liver with blood from the dog’s own
vascular system through an extracorporeal circuit
driven by a pump with positive action valves. It
permits the increase of flow even in damaged
livers. In normal dogs arterial autoperfusion of the
liver accelerated the fall of plasma levels of thio-
pental. Detoxication of thiopental, retarded in
Eck fistula dogs, became normal after arterial
autoperfusion. The rate of detoxication of thio-
pental was decreased in dogs by an Eck fistula with
subsequent (30 hr.) ligation of the common hepatic
artery, but was restored to normal by arterial or

me 16

LW,
pt. of
eland,

ase in
ration
zyme
on by
slices,
splace
ylase,
imilar
moge-
ted in
which
rylase
hilled
e was
After
of the
agne-
pres-
on. In
phos-
‘imes,
he ho-
When
added
d and
ed by
‘ylase
of the
ieved
rt /50
even

sr on
1. M.
p, W.
LANG
ology,

ation
thio-
d has
oper-
3 Own
ircuit
es. It
raged
of the
thio-
ed in
terial
thio-
with
patic
ial or

March 1956

venous autoperfusion of the liver. The rate of
detoxication of thiopental sodium depends largely
on the amount of blood flowing through the liver,
but also on the state of the parenchyma. Histo-
chemical analysis of biopsies from ischemic
necrotic livers showed a great increase in non-
acidic fat. The fat accumulated characteristically
in the necrotic or surviving cells situated at the
circulatory periphery, i.e. zone 3 and zone 2 of the
liver acinus, especially in the row of surviving
cells adjacent to the necrotic areas. Arterial auto-
perfusion made this fat almost disappear. Venous
autoperfusion of the ischemic necrotic livers had a
similar effect to a lesser degree.

1093. Quantitative determination of C-re-
active protein by complement fixation.
Maurice M. Rapport AND LISELOTTE GRaF.*
Div. of Labs. and Research, New York State Dept.
of Health, Div. of Exptl. Pathology, Sloan-Ketter-
ing Inst. for Cancer Research, and Sloan-Kettering
Div., Cornell Univ. Med. College, New York
City.

In order to investigate the relationship of serum
levels of C-reactive protein to type and stage of
malignant disease, a complement fixation method
has been developed which yields an objective
numerical index representing the titer. The prin-
ciples involved in this method include the removal
of residual anticomplementary complexes from the
absorbed antibody reagent (CRPA-Schieffelin),
the selection of an antiserum dilution offering the
most economical quantity of antibody to maintain
excess for the quantity of antigen to be measured,
the use of a standard antigen preparation to con-
trol variation in reagents and fixability of comple-
ment, and the choice of the degree of sensitivity
most suitable for a given purpose. The sensitivity
of the method adopted was set to conform to the
results obtained with capillary precipitation tech-
nique in the lower range of serum CRP. Themethod
involves an antigen titration (2-fold serial dilu-
tions of the patient’s serum starting at 1:10) in
the presence of excess antibody and four 50%
hemolytic units of complement, with graphical
estimation from a plot of log dilution vs. logit
hemolysis of the quantity of antigen which pro-
duces exactly 50% hemolysis. The results indicate
that the range of CRP levels extends from less than
0.002 mg/cc to more than 0.300 mg/ce in various
cases of cancer.

1094. Formation of succinic dehydrogenase
and cytochrome oxidase during aeration of
anaerobically grown yeast. CAROLINE Raut
AND JoaN Burman (introduced by Puruip G.
Srans.y). Detroit Inst. of Cancer Research and
Wayne Univ. College of Medicine, Detroit, Mich.
Increase in activity of succinic dehydrogenase

and of cytochrome oxidase on aeration of anaer-

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

335

obically grown yeast has been studied. Subcel-
lular particles from broken cells, which were spun
down in the ultracentrifuge, were assayed for ac-
tivity. The activities of both these enzymes in-
crease up to over 60-80 times during the course of
the adaptation. Depending on how the anaerobic
yeast was grown and the resultant state of the
cells, the increase in activity of both the enzymes
proceeds at a more or less rapid rate. The relation
between increase in enzyme activity and the mito-
chondrial content of the cells during the process of
adaptation was studied.

1095. Influence of prolactin on coenzyme A of
lactating mammary tissue. MERRILL 8. Reap
AND Ricuarp O. Moors (introduced by Joun B.
Brown). Dept. of Agricultural Biochemistry,
Ohio State Univ., Columbus.

The injection of prolactin into lactating guinea
pig mammary glands in situ causes a significant
increase in coenzyme A (CoA) concentration
within 1 hr. after injection. Aqueous homogenates
of lactating guinea pig mammary tissue (12-36 hr.
postpartum) degrade CoA at pH 8.0 at an ex-
tremely rapid rate. This degradation is inhibited
by phosphate ions and by a decrease in pu to 7.4.
CoA degradation at px 8.0 is also inhibited by the
hormone prolactin. Prolactin inhibition appears to
be due to the formation of a prolactin-CoA com-
plex which may be cleaved by crude pigeon liver
acetone powder extracts but not by more highly
purified CoA assay systems.

1096. Glucose metabolism by the alga
Ochromonas malhamensis. GEORGE H. ReEa-
ZIN, JR.* AND Martin Gripss. Dept. of Biology,
Brookhaven Natl. Lab., Upton, N. Y.

Young resting cells of O. malhamensis fermented

1 m of glucose to 1.6 m of carbon dioxide, 1.75 m

of ethanol and 0.18 m of lactic acid. When glu-

cose-1-C' (specific activity 13) was fermented,
the tracer was found exclusively in the methyl
carbon of ethanol (sp. ac. 40). Glucose-6-C™
gave similar data. When glucose was respired in
the presence of arsenite (2.5 X 107% M) pyruvic
and lactic acids accumulated. Pyruvie acid
formed during the oxidation of glucose-2-C'*
(sp. ac. 17.5) had tracer exclusively in the alpha
carbon (sp. ac. 52). Pyruvic acid formed aero-
bically from glucose-1-C'4 was labeled in the
methyl carbon. The carbon dioxide arising during
the oxidation of glucose was unlabeled. These
data indicate that the Embden-Meyerhof path-
way is the major, if not the sole means, of glucose
dissimilation by this organism. (Supported by the
Atomic Energy Commission.)

1097. Effects of treating estrogens with zinc
and hydrochloric acid. Lois REcKERS* AND
Puitie A. Katzman. Dept. of Biochemistry,
St. Louis Univ. School of Medicine, St. Louis, Mo.

336

A study of the effect of heating aqueous solu-
tions of free estrogens with HC] and zinc dust has
been initiated as part of an investigation to eluci-
date the reactions which have been reported to
give rise to unexpectedly high estrogen values
when certain urines are subjected to such treat-
ment. In dilute solution (<10 y of estrogen/ml)
estrone and estradiol were inactivated biologically
and no longer responded to Kober’s reagent.
Estriol lost about 60% of its potency and its
Kober chromogenicity to a lesser extent. Under
these conditions, biological activity or Kober
reactive material was not obtained in the phenolic
fractions from estriol glucuronide. With higher
concentrations of the estrogens (50-100 y of
estrogen/ml), which permitted weighing of the
fractions, about 20% of estriol and estradiol was
converted to material which appeared in the
neutral fractions and which had little biological
activity and a low absorption at 540 my in the
Kober reaction. The material remaining in the
phenolic fractions exhibited lower biological
activity than that of the original respective
estrogens. A larger portion of estrone was con-
verted to material recovered from the neutral
fraction which, on the basis of melting point and
infrared spectrum, was identical with 17-deoxy-
estrone prepared from estrone by a modified Wolff-
Kishner reaction. The phenolic fraction appeared
to be unchanged estrone.

1098. Enzymatic release of nonprotein sulf-
hydryl from tissue homogenates. U. D.
Reaister, ARMAND M. LaSorsa anp Davip M.
Katsuyama (introduced by Merritt N.
CamIEN). Dept. of Biochemistry, School of
Medicine, College of Med. Evangelists, Loma
Linda, Calif.

Studies in this laboratory have shown that cold
and restraint decrease the level of liver nonprotein
sulfhydryl compounds (NPSH) in rats on a stock
diet but gjgnificantly increase liver NPSH in rats
on protein-free and methionine-deficient diets.
The liver NPSH in rats maintained on a protein-
free diet for 3 days decreased from a control
average of 850 um% to 350 um%. On a stock diet
the values returned to 800 um% within 4 hr. and
increased to over 1100 um% within 14 hr. The
soluble protein sulfhydryl (PSH) showed little
change during the regeneration period. The PSH
and NPSH were measured by amperometric
titration methods. An attempt was made to de-
termine whether these rapid changes were due to
the synthesis of NPSH or to the release of SH
from liver proteins. Krimsky and Racker (J.
Biol. Chem. 198: 721, 1952) have shown that heat
or trypsin treatment of purified glyceraldehyde-3-
phosphate dehydrogenase will release glutathione
(GSH) from the protein. When liver homogenates

FEDERATION PROCEEDINGS

Volume 16

were incubated with 2-5 mg of crystalline trypsin
for 15 min., the NPSH of the stock group in-
creased from 850 um% to 1600 um%, the protein-
free group increased from 350 um% to 1300 um%,
and the 14-hr. regeneration group increased from
1100 um% to 2100 um%. The PSH after trypsin
treatment showed decreases of 400, 650 and 700
uM%, respectively. The fact that the decrease in
PSH does not account for the total increase in
NPSH after enzyme treatment suggests that part
of the NPSH released from protein was bound
through the SH group. The nature of this released
SH has been studied together with the PSH and
NPSH changes observed above. If the SH released
from protein is in the form of glutathione, then the
glutathione level in the liver must be considerably
higher than that indicated by conventional
methods.

1099. Inorganic chick growth promoting
substances from natural products. B. L,
Reip,* R. L. Svacua* ano J. R. Coucn. Depts.
of Poultry Husbandry and Biochemistry and
Nutrition, Texas A. and M. College, College
Station, Texas.

Reports from this laboratory and from Cornell
University have shown that the ash of unidentified
growth factor sources (distillers dried solubles,
dried whey and fish solubles) increased the growth
rate of chicks when supplemented to a purified
type diet or an all-vegetable protein diet. Spectro-
graphic analysis of the ash of these products has
demonstrated the presence of 23-26 elements. The
ash obtained from these products was reconsti-
tuted using reagent grade chemicals and was found
to stimulate the growth of chicks to the same
extent as did the ashed materials. Studies on the
individual elements present in the ashed products
have led to the conclusion that a portion of the
growth response may be due to the molybdenum
content. However, reconstituted ash mixtures in
which molybdenum was omitted were still active
in stimulating the growth rate of chicks. Growth
responses of around 15% have been obtained by
the feeding of distillers dried solubles ash, the
reconstituted product or 0.0126 mg molybdenum/
kg. Data have been accumulated on the influence
of mineral balance on the growth response ob-
tained with the inorganic constituents of the un-
identified factor sources.

1100. Intestinal absorption of unhydrolyzed
tripalmitin. RayMonp REISER AND JULIUS W.
DreckerT.* Texas Agricultural Exp. Station,
College Station.

A mixture of 20 mg of tripalmitin, C'4-labeled
in both the glycerol and acid moieties, and 180 mg
of triunsaturated triglycerides, was mixed in 1 gm
of a fat-free ration and fed to a rat with a thoracic

Reid fee feed. Oo feet | (lee ee

pre

ad
pl

ust
br
ciy

ume 16

rypsin
up in-
‘otein-
| uM,
1 from
rypsin
ad 700
ase in
ase in
it part
bound
leased
H and
leased
en the
erably
tional

oting
B. L,
Depis.
y and
ollege

ornell
tified
ubles,
rowth
irified
ectro-
ts has
:. The
onsti-
found
same
nn. the
ducts
f the
enum
res in
wctive
rowth
2d by
, the
num/
uence
e ob-
e un-

lyzed
1s W.
ution,

beled
30 mg

1 gm
racic

March 1956

duct cannula. Lymph was collected for 9 hr. A
second meal was fed and the lymph collected for
6 hr. It was found that in the 2 periods, respec-
tively, 3.3 and 3.2% as much of the labeled glycerol
appeared in the lymph saturated triglyceride as
would have appeared there had all the labeled
lymph acid been absorbed unhydrolyzed. It was
concluded, therefore, that only 3.3 and 3.2% of
the tripalmitin was absorbed unhydrolyzed.

1101. Anaerobic carbon dioxide production
from bicarbonate with sugar utilization in
homogenates of transplanted human and
animal tumors. Morra Davison REYNOLDS,
EuGcEeNnE L. Brvurn, Henry M. LEMoN AND
Mary Louise TurN»eR (introduced by J. M.
Looney). Depts. of Biochemistry and Medicine,
Boston Univ. School of Medicine, Boston, Mass.
Homogenates were made of the Lutz methyl-

cholanthrene-induced hamster sarcoma, and of 2

transplanted human carcinomas originally sup-

plied by Dr. Bradford Patterson: a parotid tumor

(Deac. I) and a thyroid tumor (Greene). All had

been serially passed in the hamster cheek pouch

for many generations. The 3 tumors gave similar
results in the following tests. Homogenates were

incubated for 1 hr. at 37° under 95% Ne and 5%

CO. in the fortified medium recommended by

LePage (J. Biol. Chem. 176: 1009, 1948). The COz

evolved by acid from the bicarbonate in the

medium was measured manometrically, and found
to exceed by 10% the theoretical amount calcu-
lated from the disappearance of glucose. Substitu-
tion of 0.01 m mannose for 0.01 m glucose caused
no change in rate of CO production. Compared to
the rate in simultaneous controls with glucose as

substrate, D-fructose was metabolized 74 to 100%;

2-deoxy-p-glucose approximately 95%; p-galactose

33-37%. The following compounds were not metab-

olized and did not inhibit glucose metabolism

when present in «quimolar concentration: D-ribose,

L-arabinose, D-arabinose, D-xylose, p-lyxose,
L-fucose, D-rhamnose, and p-mannitol.
1102. Determination of acid aminopoly-

saccharides in human urine. CLaytTon RicH

AND Nicota D1 Ferrante (introduced by

Dominic D. Dz1ew1aTKowsk1). Rockefeller Inst.

for Med. Research, New York City.

Acid aminopolysaccharides of urine were
precipitated as complexes with cetyl trimethyl
ammonium bromide. Conditions under which
added skeletal chondroitin sulfate could be com-
pletely recovered from urine were determined and
used: To 30 ml of urine at pH 5, 1 ml of a 2.5%
aqueous solution of cetyl trimethyl ammonium
bromide was added. After 15 hr. at 2° the pre-
cipitate was isolated by centrifugation and washed
with 95% ethanol saturated with sodium chloride

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

337

thereby removing the precipitant. The residue was
then dissolved in water and its glucuronic acid
content determined. The material in solution when
mixed with a toluidine blue solution produced a
metachromasia, it did not pass through a cello-
phane membrane on dialysis against water, and it
resembled skeletal chondroitin sulfate in its
electrophoretic mobility on paper and in chroma-
tography on paper. Incubation of this material
with crystalline trypsin did not produce dialyzable
glucuronic acid but subsequent treatment with
testicular hyaluronidase did depolymerize it. Its
excretion in the urine of 12 normal adults was the
equivalent of 1.7-5.1 mg of glucuronic acid per
24 hr.

1103. Mechanism of action of muscle triose
phosphate isomerase. 8S. V. RrEDER ANp I. A.
RoseE (introduced by 8. Stmmonps). Dept. of
Biochemistry, Yale Univ., New Haven, Conn.
Triose phosphate isomerase, crystallized from

muscle, was incubated with dihydroxyacetone

phosphate (DHAP) in tritiated water. The DHAP
was recovered by chromatography on Dowex-l

Cl-, and was found to have acquired tritium.

Under conditions of high enzyme concentration

and prolonged incubation the specific activity of

the. DHAP (cpm/um) was found to approach
closely but not to exceed the specific activity of
the hydrogen (cpm/yatom) of the water. If the
enzyme is omitted, glyceraldehyde-3-phosphate
and DHAP do not acquire tritium that is not
washed out in the isolation procedure. These
results imply the formation by the enzyme of an
intermediate which is in equilibrium with tritium
of the water. The finding that the specific activity
of the DHAP does not exceed that of the hydrogen
of the medium indicates that the exchange re-
action is stereospecific for one of the four carbon-
bound hydrogens of DHAP. Previous work from
this laboratory (Rose anp RiepER, J. Am.
Chem. Soc. 77: 5764, 1955) has indicated that
muscle aldolase causes the stereospecific exchange
of one of the hydrogens bound to the carbinol
carbon of DHAP. In order to determine whether
this same hydrogen is the one labilized by isomer-
ase, DHAP labeled with tritium by aldolase was
incubated with isomerase, and DHAP labeled with
tritium by isomerase was incubated with aldolase.

No significant lowering of the specific activity of

DHAP was found in either case. This indicates

that the exchange catalyzed by the two enzymes

involves different hydrogens.

1104. Interrelations among pyridoxal, alkali
metal ions and amino acids in active trans-
port. T. R. Riaes anp H. N. CHRISTENSEN.
Dept. of Biological Chemistry, Univ. of Michigan,
Ann Arbor.

A study of interrelationships between amino

338

acid and alkali metal transport has continued in
two directions. First the effect of varying the Kt
levels has been explored more closely. The degree
of concentration of glycine by Ehrlich ascites-
tumor cells was halved by decreasing the final
extracellular K* level of the medium to 1 mm.
The decrease was linear in the range 3 to 1 mm.
No K* was included in the medium of these experi-
ments, the extracellular K* arising from outflux
from the cells. The final level of extracellular Kt
could be predicted and controlled by varying the
volume of K*-free suspending fluid used. A plot
of the log [K+] against the log of the ratio of the
volume of the extracellular fluid to the cellular
volume, which was linear, was used for this pre-
diction. The results suggest that extracellular K
is necessary for glycine uptake. Furthermore there
was no stimulation of glycine uptake by pyridoxal
at 1 mm extracellular K*. The largest effect of
pyridoxal on glycine uptake was obtained at 15
mm Kt. The consequence is that the glycine
transport system tolerates a higher K* level when
pyridoxal is present. Secondly, the nature of the
differential reaction between pyridoxal and alkali
metals (Science 122: 1087, 1955) was studied by
titration and spectrophotometry. Factors in the
differentiation between Na* and Kt appear to be
a) the free state of the 5-hydroxymethyl group,
b) the state of hydration of the carbonyl group,
and c) hemiacetal formation between the two
groups.

1105. Sulfate activation. PHILLIPS W. RopBins*
AND Fritz LipmMann. Biochemical Research Lab.,

Massachusetts General Hosp., and Dept. of
Biological Chemistry, Harvard Med. School,
Boston.

The recently reported work on sulfate activa-
tion (H. Hitz anp F. Lipmann, Proc. Nat. Acad.
Sc. 41: 880, 1955) was continued. Fractionation of
the sulfate-nitrophenol transfer system in liver
extracts, yielded an acceptor enzyme freed of
sulfate-activating enzyme. This fraction was used
for assay of active sulfate produced by the sulfate-
activating fraction of the same extract. It appears
that active sulfate, preliminarily characterized as
an adeny] sulfate, is relatively stable at px 4-5.
(Supported by a Life Insurance Med. Research
grant.)

1106. Mucopolysaccharide formation in scor-
butic repair tissue. WM. vAN B. RoBERTSON,
JANE THURLOW* AND HusBert Hinps.* College
of Medicine, Univ. of Vermont, Burlington.

The repair tissue which forms in guinea pigs
deprived of ascorbic acid and which contains very
little collagen (J. Biol. Chem. 201: 689, 1953) was
found to contain about five times as much amino-
mucopolysaccharide as the collagenous repair
tissue from adequately fed guinea pigs. The data

FEDERATION PROCEEDINGS

Volume 1§

suggest that there is an excess formation of
polysaccharides in granulation tissue during
scurvy and not simply a failure to remove nor-
mally forming polysaccharides. The major muco-
polysaccharide which accumulates in the scorbuti¢
granulation tissue was isolated and identified as
hyaluronate.

1107. Influence of mineral elements and pH
upon the hatching of golden nematode
(Heterodera rostochiensis Wollenweber)
larvae. TREVOR Rospinson AND A. L. NEAL,
(introduced by H. H. Wiuuiams). Dept. of Bio-
chemistry and Nutrition, Cornell Univ., Ithaca,
No?

During studies undertaken to isolate the golden
nematode hatching stimulant which is excreted
by the roots of potato and tomato, it became
apparent that mineral ions and pu were influencing
the hatch. The cations and strong anions were
removed from tomato plant leachings by means of
Amberlite IR-120 and IR-4B ion exchange resins,
The effluent possessed about one-half of the
activity of the original leachings. Complete ac-
tivity could be regained by adding back a HCl
eluate from the IR-120 column. The cation
chlorides themselves were not significantly active
in the absence of the effluent. The chlorides of Ca,
Mg, Na and K (450, 27.4, 12.5, 9.3 ug/mi respec-
tively) could replace the eluate and all were
essential for maximum activity of the cation-free
concentrates of the natural stimulant. Of these
cations potassium appeared to influence hatch to
the greatest extent. A significant increase in hatch
occurred when a solution of the above salts was
adjusted to px 2.5 with HCI, malic or citric acid.
The hatch induced under these conditions was
considerably less than that observed with the
natural stimulant plus the cations. Trace amounts
of Zn, Cd, Co, Cu, Mg, Fe and B had no effect
upon the hatching of larvae. However, at levels
above 0.5 ug/ml Cd and Zn were inhibitory. The
anions eluted from the IR-4B column with NH,OH
produced no stimulatory effect.

1108. 8-Hydroxyisobutyric dehydrogenase.
Wixi1am G. Rosrnson (introduced by Hatvor
N. CHRISTENSEN). Dept. of Biological Chemistry,
Univ. of Michigan, Ann Arbor.

In the course of studies on valine and iso-
butyrate metabolism a new DPN-linked de-
hydrogenase catalyzing the oxidation of
B-hydroxyisobutyrate has been found in micro-
organisms and animal tissues. The reaction
product, methylmalonaldehydate, has been
identified chromatographically and has_ been
synthesized and shown to be reduced to B-hydroxy-
isobutyrate in the presence of DPNH and the
dehydrogenase. The enzyme exhibits activity
neither with n-8-hydroxybutyrate, malate, lac-

tat
hy
B-I

pre
bee
he:
Te
br:
en
of

bu
or

int
sul
th:

fro
aci
ree
agi
lak

ser
als
fer
(1.
sta
ser
bel
Ox)
Oxi
con
tio:
of
pro
ran
of |
of f

cul,
cys
spe
wit

lll

ume 1§

on of
luring
e nor-
muco-
rbuti¢
ied ag

id pH
atode
reber)
NEAL,
f Bio-
[thaca,

rolden
creted
ecame
sncing
} were
ans of
resins.
f the
te ac-
2 HCl
cation
active
of Ca,
espec-
were
n-free
these
tch to
hatch
‘8 was
: acid,
S was
h the
lounts
effect
levels
-. The
H,OH

nase,
ALVOR
nistry,

1 iso-
1 de-
1 of
nicro-
ction
been
been
lroxy-
d the
tivity
lac-

March 1956

tate, homogerine, pantoate and a variety of other
hydroxy acids in the presence of DPN, nor with
6-hydroxyisobutyrate in the presence of TPN. As
assayed by the rate of DPN reduction in the
presence of 8-hydroxyisobutyrate, the enzyme has
been demonstrated to be present in extracts of
heart, liver, kidney, Aerobacter aerogenes, and
Tetrahymena pyriformis, but to be absent from
brain and spinach extracts. The partially purified
enzyme from potassium chloride-alcohol extracts
of pig kidney is inhibited by sulfhydryl reagents,
but not by metal chelating reagents like EDTA
or citrate. Since 6-hydroxyisobutyryl CoA, an
intermediate in valine metabolism, is not a
substrate for the dehydrogenase, it is presumed
that coenzyme A is removed enzymatically prior
to dehydrogenation. The absence of coenzyme A
from the carboxy] carbon of the resulting aldehyde
acid (or further metabolites) should permit the
ready loss of this carbon as carbon dioxide, in
agreement with isotopic studies from other
laboratories.

1109. Oxidation of cysteine to cystine by
oxygen and effect of iron on reaction rate.
F. Leg Ropkey. Dept. of Biological Chemistry,
Harvard Med. School, Boston, Mass.

The oxidation of cysteine was studied by
measuring the rate and extent of oxygen con-
sumption. Initial rates were determined as a
function of the cysteine concentration and the
pH of the solution. The maximum rate occurred at
pH 8.75 with 1.3 X 107? m cysteine in the absence
of added metals. Negligible oxidation was ob-
served below pH 7.5 and above 10.0. A study was
also made of the oxidation in the presence of
ferrous iron. Holding the concentration of cysteine
(1.3 X 10-2 m) and ferrous ion (2 X 10~® Mm) con-
stant and varying the pH, a maximum rate was ob-
served at pH 8.5. Very little oxygen was utilized
below pH 6.0 or above 11.0. A maximum of 1 of
oxygen was consumed for every 4 M of cysteine
oxidized. Holding the concentration of cysteine
constant (1.8 X 107? Mm) at pu 8.5, the concentra-
tion of ferrous ion was varied. The initial rate
of oxygen consumption was found to be directly
proportional to the concentration of iron in the
range of 1 X 10-* to 1 X 10° molar. Titrations
of cysteine alone and of cysteine in the presence
of ferrous iron were performed with tetraethylam-
monium hydroxide. These data were used to cal-
culate the stability constants of the various iron-
cysteine complexes. The concentration of the
specific iron-cysteine chelates will be compared
with the observed oxidation rates.

1110. N-glucosylglycine-requiring mutants of
Escherichia coli. DExTER RoGERs (introduced
by Josrru P. CHANDLER). Dept. of Biological
Chemistry, Univ. of Michigan, Ann Arbor.

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

339

Fourteen biochemical mutants of Escherichia
coli OSC have been isolated which can be arranged
into 2 broad categories: group A, incompletely
blocked mutants responding to glucosylglycine, its
Amadori rearrangement product or yeast extract;
and group B, completely blocked mutants re-
sponding to yeast extract only. Culture filtrates
of the group B organisms stimulate the group A
organisms, and mixed cultures of groups A and B
organisms grow symbiotically in the minimal
medium. With the »id of chromatography, it was
found that the growp A active material accu-
mulating in the medium during growth of a group
B organism is not identical with the factor in
yeast extract which is necessary for growth of
both the growps A and B organisms. This inter-
mediate appears to be acidic and more polar than
the yeast extract factor. There exists evidence for
still other factors. These may be degradative
products or metabolic intermediates. The activity
has been concentrated to a tan powder, and it is
interesting to note that the accumulated inter-
mediate when autoclaved with the mineral supple-
ment of the medium will stimulate the group B
organism that produced it.

1111. Enzymatic reduction of the double bond
of Ring A of certain steroids. Epwarp L.
RonGonE,* BERNADETTE C. BockuiaGeE,* D. R.
STRENGTH* AND Epwarp A. Dorsy. Dept. of
Biochemistry, St. Louis Univ. School of Medicine,
St. Louis, Mo.

Solutions of commercial preparations of bovine
blood albumin contain an enzyme which catalyzes
the reduction of the double bond of progesterone
and of A‘-androstene-3, 17-dione to yield pregnane-
dione and etiocholane-3,17-dione, respectively.
The products obtained from the incubation
mixture were identified by melting points, mixed
melting points, optical rotation and infrared
spectra. Incubation of cortisone with 5% albumin
solutions containing 3% ethanol resulted in the
loss of absorption at 238 my with no effect on the
a-ketol side chain as determined by the Porter
and Silber reaction. Dialysis of the albumin caused
loss of the reductive action. Activity was restored
by the addition of TPN and citrate or isocitrate.
TPN could not be replaced by DPN. The optimum
conditions for the reduction were at pH 6.4, 37.5°C
and a cortisone concentration of about 10-4 m.

1112. Investigations on inhibition of L.
bifidus enzyme. CATHARINE S. RosE AND
Paut Gyérey. Dept. of Pediatrics, Univ. of
Pennsylvania, Philadelphia.

Activity as an essential growth factor for L.
bifidus var. Pennsylvanicus is demonstrated by a
number of compounds of high or low molecular
weight, but all containing N-acetyl-p-glucos-
amine. A cell-free extract of L. bifidus var.

340

Pennsylvanicus liberates N-acetyl-p-glucosamine
from these compounds with concomitant loss of
bifidus factor activity. Certain sugars inhibit the
action of the L. bifidus enzyme on mucopoly-
saccharides of human milk or hog gastric mucin.
p-galactose, N-acetyl-p-glucosamine and L-fucose
had marked effect at a 4% level. With human milk
and nondialyzable active fractions from human
milk L-fucose was the most effective of the sugars,
while the gastric mucin L-fucose was less active
than p-galactose or N-acetyl-p-glucosamine.
Other sugars including p-glucose, p-fructose,
L-arabinose and D-mannose had essentially no
effect. The specificity of the effect of L-fucose on
the bifidus factor activity of human milk was
supported by in vivo tests. L-fucose at a level of
1 mg/ml markedly inhibited the growth of L.
bifidus var. Pennsylvanicus when bifidus factor
was supplied as human milk or purified fractions
from human milk, but not when other forms of
bifidus factor were used. The inhibition was not
complete and the degree of inhibition was not
increased appreciably by larger amounts of fucose.

1113. Preparation and metabolic fate of
crystalline glucosamine-phosphate. Sau
Roseman. Rackham Arthritis Research Unit and
Dept. of Biological Chemistry, Univ. of Michigan,
Ann Arbor.

The polyphosphoric acid method for the prepa-
ration of glucose-6-phosphate (G-6-P) has recently
been applied by other workers to glucosamine
(Gm) and yielded a crude, amorphous barium salt.
Modification of the precedures gave a stable,
crystalline product which is apparently glu-
cosamine-phosphate (Gm-P) per se, probably
glucosamine-6-phosphate. It was also possible to
isolate crystalline material from the hexokinase
reaction (glucosamine and ATP). Comparison of
the enzymatic and synthetic products is in
progress. The metabolic fate of the synthetic
product*® (Gm-P) was studied using extracts
obtained from Escherichia coli. The extracts were
incubated with various phosphorylated sugars in
the presence and absence of TPN. A rapid re-
duction of TPN occurred in the presence of
Gm-P, G-6-P or fructose-6-phosphate (F-6-P).
Inhibition and rate studies suggested that the
oxidation of Gm-P (concomitant reduction of
TPN) proceeded via the hexose-phosphates. This
concept was tested by incubating the mixtures
without TPN. The Gm-P was rapidly utilized and
formed a mixture of fructose, F-6-P,: glucose,
G-6-P, ammonia, and traces of Gm. The total
ammonia, hexose, and hexose-phosphates was
essentially equivalent to the Gm-P consumed.
Early in the reaction, the predominant products
are F-6-P and G-6-P (Soopaxk, M., Bact. Proc.
131, 1955). We have recently reported that the

FEDERATION PROCEEDINGS

Volume 16

synthesis of Gm-P requires F-6-P (Neurospora
extracts). Nevertheless, in the reverse reaction
(E. coli extracts and Gm-6-P), the ‘first’ product
(G-6-P or F-6-P) is unknown. The conversion of
Gm-P to hexose-P is the rate-limiting step in the
sequence.

1114. Transformation of cholesterol-3d,4C%
to coprostanol; location of deuterium in
coprostanol. R. S. RosENFELD,* LEon HELL-
MAN* AND T. F. GALLAGHER. Sloan-Kettering
Inst. for Cancer Research, New York City.
Recent work from this laboratory has dealt with

the biochemical mechanisms by which the con-
version of cholesterol to coprostanol is accom-
plished in human subjects and in incubation
experiments. When cholesterol-4-C' and cho-
lestenone-4-C™“ were separately incubated with
human feces, approximately 40% of each was
converted to coprostanol. No cholestenone could
be identified in the mixture after the cholesterol
incubation. In the cholestenone incubation, no
trace of labeled cholesterol was found. The major
portion of the radioactivity was present as un-
reacted cholestenone, other than that converted
to coprostanol. These data suggest that although
cholestenone can be reduced in the presence of
feces, it is not necessarily a normal intermediate
in the formation of coprostanol. In similar incuba-
tion studies, cholesterol-3d,4C* was transformed
to coprostanol which contained both isotopes.
The resultant coprostanol contained deuterium at
other positions in the molecule in addition to
C-3. By stepwise removal of hydrogen from C-3,
C-5, and C-6 through the intermediates coprosta-
none and 48-bromocoprostanone, A‘-cholestenone
was obtained devoid of deuterium. Therefore the
deuterium in the coprostanol could only have been
at C-3, C-5 and C-6 and no other site in the
molecule was altered during the biological re-
duction of cholesterol.

1115. Biliary excretion of alkaline phospha-
tase in the rat. Ortro RosENTHAL, GIANCARLO
CasTIGLIONI,* ELENA ARMOCIDA* AND JOHN
Hosart.* Harrison Dept. of Surgical Research,
Schools of Medicine, Univ. of Pennsylvania,
Philadelphia.

We have previously reported that the activity
levels of the cyanide-sensitive alkaline phos-
phatase of rat liver are related to food absorption.
Since the plasma phosphatase levels of the rat are
known to depend on fat absorption the possibility
was considered that plasma phosphatase was taken
up by the liver and either destroyed or excreted
via the bile. To test the latter alternative phos-
phatase assays were done upon plasma and bile
of rats with draining bile fistulae during alternate
5-6 day periods on fat-free and on 6% cod-liver

te’

lume 18

rospora
eaction
oroduet
‘sion of
» in the

3d ,4C%
um in

HELL-
ettering

lt with
1e con-
accom-
ibation
d cho-
d with
ch was
9 could
lesterol
ion, no
» major
as un-
verted
though
ance of
nediate
incuba-
formed
otopes.
‘ium at
jion to
m C-3,
prosta-
tenone
ore the
ve been
in the
cal re-

»spha-
NCARLO

JOHN
search,
ilvania,

ctivity

phos-
rption.
rat are
sibility
3 taken
ccreted
» phos-
ad bile
bernate
d-liver

March 1956

oil plus 0.47% cholic acid-containing diets. The
cholic acid supplement achieved about 65%
absorption of the dietary fat. Shift from the fat-
free to the fat-containing diet resulted in average
phosphatase increases from 20 to 56 and 4.7 to 8.6
Bodansky v/100 ml in plasma and bile respectively.
The daily biliary phosphatase excretion rose from
0.5 to 1.3 vu. The experiments show that both
plasma phosphatase levels and concentration and
output of biliary phosphatase are augmented by
intestinal fat absorption. However, biliary ex-
cretion was small in comparison to the amount of
enzyme known to be released into the circulation
during fat absorption. This fact together with the
absence of a definite quantitative relationship
between plasma and bile levels of the enzyme
point to two independent effects of fat absorption
rather than to biliary elimination of plasma
phosphatase. (Supported in part by Army Con-
tract DA-49-007-MD-143 and a grant from the
American Cancer Society.)

1116. Interaction of yttrium compounds with
serum proteins. Brtrry Rosorr,* RvutTH
Lewin,* Hrram E. Hart* anp Kurt G. STERN.
Div. of Neoplastic Diseases, Montefiore Hosp.,
New York City, and Dept. of Chemistry, Poly-
technic Inst., Brooklyn, N.Y.

As part of a research program dealing with the
biological behavior of stable and radioactive rare
earths, including yttrium, in vitro experiments on
the binding of yttrium in ionic and chelated form
to serum proteins have been performed. Employ-
ing Y°° and Y®! as tracers yttrium chloride and a
series of yttrium chelates with ethylenediamine-
tetraacetic acid, isopropylenediaminetetraacetic
acid, N-hydroxyethylethylenediaminetriacetic
acid, nitrilotriacetic acid, 6B-hydroxyethyliminodi-
acetic acid, N,N’-dihydroxyethylglycine, and
iminodiacetic acid were added to whole serum and
serum protein fractions including serum albumin.
Equilibrium dialysis of solutions of albumin
against YC]; showed that Y-protein binding rises
with increasing pH (4-6) an increasing relative
Y** concentrations. At higher Y*** concentra-
tions more than 15 M of yttrium combined with 1 mu
of albumin. The binding sites for yttrium in serum
are being investigated by means of zone (paper
and starch block) electrophoresis. An appreciable
fraction of yttrium added as YCl; migrated at
mobilities in the globulin range. Another fraction
exhibited higher mobility than that of the albumin
zone. A close correlation between Y-chelate
stability (log Ki) and transfer of yttrium from the
chelate to the serum proteins was demonstrated
in pressure filtration studies. For Y-ethylenedi-
aminetetraacetate (log Ki ~ 14), about 90% of
yttrium was filtrable, while at low concentrations
of Y-8-hydroxyethyliminodiacetate (log Ki ~ 8.6),
only 1% passed through the membrane.

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

341

1117. Purification and partial characteriza-
tion of ribonucleases of rat liver mito-
chondria. Jay 8. Rots (introduced by JospPH
S. Hepsurn). Div. of Biological Chemistry,
Hahnemann Med. College, Philadelphia, Pa.

Rat liver mitochondria were prepared by dif-
ferential centrifugation in sucrose. Two methods
were used to solubilize the enzymes: 1) treatment
with 0.25 m H2SO, at 0° for 24 hr., 2) addition of
sodium deoxycholate followed by salt fractiona-
tion. Incubation with acid destroys acid ribo-
nuclease (RNase) activity (measured at pH 5.8)
but has no effect on alkaline RNase (measured at
pH 7.8). Since acid treatment precipitates approxi-
mately 95% of the mitochondrial N, alkaline
RNase specific activity is increased 20-fold based
on the original mitochondrial preparation. 0.2%
deoxycholate solubilizes both mitochondrial
RNases without altering the activity of the
alkaline enzyme, but the acid enzyme is partially
destroyed. By careful treatment, however, most
of the acid RNase activity may be retained
throughout the purification process. Final deoxy-
cholate treated preparations have 25- and 5-times
the activity of the original homogenate and
separated mitochondria, respectively; recovery of
the alkaline enzyme is approximately 50%. The
effect of various agents on RNase activity of
separated mitochondria was determined. Tris
buffer activates alkaline RNase, while phosphate
and ABC are somewhat inhibitory; with acid
RNase, veronal-acetate gives maximum activity
and citrate, acetate and ABC are inhibitory.
Neither enzyme is appreciably affected by 4 X
10-* m p-chloromercuribenzoate. These results,
and others to be presented, suggest that alkaline
RNase of liver is similar to, or identical with,
crystalline pancreatic RNase, while acid RNase is
quite different and may be a phosphodiesterase of
more general specificity than the alkaline enzyme.
(Supported by PHS Grant C-2312).

1118. Eluted antibodies. Kart L. Rota anp
ABRAHAM M. FrRumiIn (introduced by CrEcrLia
RriEGEL). Dept. of Research, Southern Div.,
Albert Einstein Med. Center, Philadelphia, Pa.
Antibody eluates were prepared from the RBC

of 27 patients with acquired hemolytic anemia.

Direct Coombs’ titers of patients’ cells and of

normal cells coated with these eluates were

established, together with eluate dilution titers
and Coombs’ serum-eluate equilibrium ratios.

These ratios were found by subjecting RBC

coated with the highest still reactive eluate dilu-

tions to decreasing dilutions of Coombs’ serum;
or by mixing the 2 factors in varying proportions,
and testing for residual Coombs’ serum or eluate
activity. RBC used in all these experiments were
freshly obtained from the same donor. Similarly,

342

the Coombs’ serum pool was identical throughout.
The results of both methods compared satis-
factorily. Considering the eluate-Coombs’ serum
reaction as an ‘antigen-antibody’ union, equilib-
rium ratios were incorporated into an equation
describing a precipitin reaction. An attempt was
made to correlate the data by assuming that
eluates from different patients were but dilutions
of each other. While results obtained justified the
above assumption, several distinct eluate groups
could be differentiated, each possessing its indi-
vidual curve. The possible relationship of these
observations to antibody avidity will be discussed,
together with reasons prohibiting the use of ad-
sorbtion experiments for antibody N (max.)
determinations. (Supported by Grant #* H-1919C
from the Natl. Insts. of Health and by a Fellow-
ship from the Joel I. Wagman Memorial Fndn.,
Philadelphia, Pa.)

1119. Amino acid metabolism in human blood
cells. GEoRGE RovusER, BoHDAN JELINEK AND
A. J. SAMUELS (introduced by D. SrmonseEn).
Depts. of Biochemistry and Medicine, Med.
Research Inst., City of Hope Med. Center, Duarte,
Calif.

A study of the free amino acids of blood plasma,
leukocytes and erythrocytes has shown that
variations in plasma free amino acids arising
spontaneously or induced by injection of amino
acids are reflected in changes in the cells. Each
morphologically distinct cell type has a charac-
teristic pattern of free amino acids. Maturation of
myelocytes to mature neutrophilic leukocytes is
associated with decreases in glutamine, gluta-
thione and ethanolamine-O-phosphate and in-
creases in cysteine and cysteinylglycine. Myelo-
cytes and myeloblasts have similar amino acid
patterns, somewhat smaller amounts of glutamine,
alanine and aspartic acid being found in myelo-
blasts. Little difference was noted in small,
medium nd large lymphocytes. Lymphoblasts
differ from more mature lymphocytes chiefly in
having less glutamine, alanine and aspartic acid
and more glutamic acid and ethanolamine-O-
phosphate. Lymphoblasts and myeloblasts, al-
though similar in pattern, can be distinguished by
differences in the concentration of glutamic acid,
ethanolamine-O-phosphate and _ glycerylphos-
phorylethanolamine. Evidence for the presence of
thetranspeptidationsystem between glutamine and
glutathione described by Hanes et al. (Nature 166:
288, 1950) in kidney and pancreas has been ob-
tained for human lymphocytes, granulocytes,
platelets and erythrocytes (normal and in leu-
kemia) and dog leukocytes. Resynthesis of
glutathione has been observed during aerobic and
anaerobic incubation.

FEDERATION PROCEEDINGS

Volume 16

1120. Unit particle lengths of strains of
tobacco mosaic virus. JOHN W. ROWEN AND
Wiiuiam Grnoza (introduced by Norman §,
MacDonatp). Atomic Energy Project, Univ. of
California School of Medicine, Los Angeles.
Eight strains of purified tobacco mosaic virus

were studied by means of streaming birefringence,
sedimentation and electron microscopy. Ex-
tinction angle and birefringence-gradient curves
were obtained of all strains in several solvents
over appropriate concentration ranges. All strains
were duodisperse. However it was feasible to make
unit length comparisons in the duodisperse state.
It was possible to compute the extinction angle
values, as a function of gradient, from relatively
small numbers of lengths obtained from the
electron micrographs. The calculated values were
in good agreement with values directly observed
in the streaming birefringence apparatus. The unit
lengths of all strains fell in the range of 3,350+-250
A in good agreement with corrected electron
microscope lengths and recently reported electric
birefringence values obtained on the common
strain. It was noted that the optical factors of
some of the strains differed from the value ob-
tained with the common strain. These differences
are discussed in the light of the possibility that
they may be related to differences in amino acid
and/or nucleotide sequences.

1121. Biosynthesis of 8-hydroxy-Smethylglu-
taric acid (HMG). Harry Rupney. Dept. of
Biochemistry, Western Reserve Univ. Med.
School, Cleveland, Ohio.

A soluble rat liver microsome preparation has
been obtained which synthesizes HMG, pre-
sumably as the CoA derivative, from acetate, ATP
and CoA, or from acetyl CoA. It is almost free of
the enzyme which forms hydroxyisovaleric acid,
but contains the acetoacetate activating enzyme.
Hitherto it has not been established whether free
acetoacetate or acetoacetyl CoA condenses with
acetyl CoA to form HMG. The following experi-
ments with labeled acyl CoA derivatives indicate
that acetoacetyl CoA is the reacting moiety.
When 4 um of C™ acetate, 3 um ATP and 0.2 uM of
CoA were incubated, addition of 10 um of aceto-
acetate reduced the incorporation of C'* into
HMG 30% below the control value. This decrease
remained unchanged when the amount of aceto-
acetate was raised to 30 or even 100 um. In this
and subsequent experiments, the extent of isotope
incorporation was measured as total counts in
HMG obtained after alkaline hydrolysis of acyl
CoA derivatives. When C™ acetyl CoA was the
substrate, unlabeled acetoacetate had no effect
on the level of C!4 in HMG, and conversely when
unlabeled acetyl CoA and C" acetoacetate were

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March 1956

incubated,.no radioactivity was detected in HMG
unless ATP was also present. Finally when only
0.2 um of acetoacetyl CoA and 0.3 um of C4 acetyl
CoA were incubated the isotope in HMG was
twice that obtained with C™ acetyl CoA alone.
These results support the hypothesis that acetyl
CoA and acetoacetyl CoA are the reacting moieties
in the formation of HMG. The question as to
whether a mono or di-CoA derivative of HMG is
formed is being investigated.

1122. Synthesis and metabolism of L-ascorbic
acid-2,3,4,5,6-C"4%. S. L. Rupourr,* R. R.
BEcKER* AanpD C. G. Kina. Dept. of Chemistry,
Columbia Univ., New York City.

L-ascorbie acid-2,3,4,5,6-C' (J) was synthe-
sized in 30% yield from p-glucose-U-C™ by the
osone-cyanide method. L-xylose-U-C", the key
intermediate, was prepared from p-sorbitol-U-C
via an improved synthesis of 2,4 monobenzylidene
sorbitol. An improved procedure for the isolation
of J using Duolite A-3 (OAc”) resin was devised. I
was injected intraperitoneally into normal guinea
pigs. After 24 hr., 11-45% of the injected activity
appeared as respiratory C“Q2. and 7-8% was
excreted in the urine. About 7% of the urinary
activity was recovered as ascorbic acid. An equal
quantity was present as labeled oxalic acid, in
agreement with earlier results obtained with
L-ascorbic acid-1-C™ (/J). In contrast to the
results of earlier studies with J7, only 38-61% of
the radioactivity present in liver tissues could be
accounted for as free ascorbic acid. Glucose,
isolated from liver glycogen, was essentially
uniformly labeled and had incorporated 0.52% of
the injected activity of J. A pathway in which
ascorbic acid is degraded to 3-carbon fragments
from positions 4, 5 and 6 which may enter the
glycolytic scheme to recombine and form uni-
formly labeled glucose is consistent with these
findings. The labeled triose may also be further
metabolized via pyruvate. Such a pathway would
account for the presence of considerable amounts
of unidentified radioactive products present in the
urine and liver of guinea pigs injected with J.

1123. Diiodotyrosine and_ triiodothyronine
deiodinase systems in liver. W. R. RusE-
GAMER AND R. B. Cuopos.* Radioisotope Unit,
VA Hosp., and Depts. of Biochemistry and
Medicine, State Univ. of New York, Syracuse.

We have shown previously (Proc. Soc. Exper.
Biol. & Med. 90: 146, 1955) that both liver and
kidney contain a mechanism for the deiodination
of diiodotyrosine (DIT). Furthermore, nephrecto-
mized dogs deiodinated DIT just as rapidly and
completely as intact animals, so that the liver
appeared to be the more important of the 2 tissues.

avery

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

343

Evidence for the enzymatic nature of this system
has now been obtained from temperature and PH
dependence studies, and fractionation of the
enzyme is proceeding at the subcellular level.
Because of the importance of liver function on
DIT metabolism in animals, studies have also been
made of the kinetics of I'*! labeled DIT and tri-
iodothyronine (T3) metabolism in patients with
cirrhosis and in normal individuals. Whole plasma
half-time values of intravenously-administered
DIT averaged 9.5 hr. and 16.6 hr. for normal and
cirrhosis patients, whereas values of 26 and 35 hr.
respectively were obtained for similar groups of
patients receiving T3. Paper chromatograms of
urine specimens revealed that relatively large
amounts of DIT and T3 pass into the urine of
cirrhosis patients whereas nearly 100% of the total
I'3! activity excreted by normal individuals ap-
peared as iodide. Larger concentrations of DIT
and T3 also remained in the circulating plasma of
cirrhosis patients. Therefore liver damage de-
creases the efficiency and completeness of DIT
and T3 deiodination as postulated, and sub-
stantiates our belief that the liver is probably the
principal site of the DIT and T3 deiodinase
systems.

1124. Metabolism of estradiol-16-C“ by
fortified enzyme systems. GIDEON RUMNEY
(introduced by Harry A. Waisman). McArdle
Memorial Lab., Univ. of Wisconsin Med. School,
Madison.

Previous studies from this laboratory by Riegel
and Mueller (J. Biol. Chem. 210: 249, 1954) have
described the formation of a protein bound
metabolite of estradiol-16-C™ in rat tissue ho-
mogenates. Further work to investigate the role
of the protein binding to the overall estrogen
action has revealed that by fractionation of mouse
liver homogenates, the enzyme effecting the
protein binding is concentrated almost entirely in
the microsome fraction and can use chemically
reduced TPN as the lone electron donor. In the
presence of oxygen, still required for the protein
binding in the microsomal system (7-9 mg dry
weight of protein), 10 y radioactive estradiol are
converted within 10 min. into the following
fractions: protein bound 15-20%, acetone-benzene
soluble 65-75% and aqueous soluble 7-10%.
Chromatography of the acetone-benzene soluble
fractions on paper, and more recently on florisil
columns with increasing concentrations of metha-
nol in benzene has revealed the presence of 5 new
metabolites, ranging in polarity from an estriol-
like fraction (T) to an estrone-like material (Z)
the intermediate metabolites being referred to as
Xi, X2 and X; in order of decreasing polarity.
The re-incubations of the isolated metabolites in

344

the microsome system have demonstrated that the
major metabolite, T, is the primary product
formed; an unstable X2 appears to result by
further metabolism of T, and X2 in turn is con-
verted to the estrone-like metabolite Z which
appears to be involved in the protein binding.

1125. Steroid 21-hydroxylation by adrenal cell
fractions. KENNETH J. Ryan (introduced by
M. Soopaxk). Med. Labs., Collis P. Huntington
Memorial Hosp. of Harvard Univ., at Massa-
chusetts General Hosp., Boston.

Investigation of the enzyme systems involved in
the conversion of 17a-hydroxyprogesterone to
17a,21-dihydroxyprogesterone (Reichstein’s Sub-
stance 8) revealed that activity was present in a
combined supernatant-microsomal system. Fresh
beef adrenals were homogenized in 2 volumes of
0.25 m sucrose with 0.05 m tris(hydroxymethy]l)-
aminomethane buffer, 0.05m KCl1,0.005 m MgCl.
and 0.005 m niacinamide. Fractionation was
accomplished by differential centrifugation, and
incubation of suitable aliquots was carried out at
pH 7, 38°C for 2 hr. in the presence of 2 mg 17-
hydroxyprogesterone, 2 mm ATP and 0.5 mm
DPN. Steroids were extracted with methylene
chloride and partitioned between 95% methanol
and hexane. The extract was assayed by the
Porter-Silber method and substance S§ identified
by Bush paper chromatography. 21-Hydroxylase
activity appeared in the supernatant after cen-
trifugation at 20,000 x g as described by Plager
and Samuels (Federation Proc. 11: 388, 1952).
There was, however, a marked loss of activity
from the supernatant if it was centrifuged at
105,000 X g. The washed and reprecipitated
microsomes had no activity alone, but recombina-
tion with the 105,000 X g supernatant restored full
activity. At optimal substrate concentration,
70-80% of added 17-hydroxyprogesterone was
21-hydroxylated. The reaction required niacin-
amide, ATP and DPN for full activity and did not
proceed anaerobically. The contributions of both
the soluble fraction and the microsomes to 21-
hydroxylation suggest a more complex system
than was anticipated from previous work. Study
of the mechanisms involved is in progress.

1126. Amino acid incorporation into stable
microsome preparations. Howarp SacuHs
AND Amos NEIDLE (introduced by H. WAELscH).
Dept. of Biochemistry, Columbia Univ., and New
York State Psychiatric Inst., New York City.
The high concentration of glutamine synthetase

in rat liver microsomes (Mc) prompted an investi-

gation of the role of glutamic acid and its thio-
esters, previously studied in this laboratory, in
amino acid incorporation. Work from other
laboratories has demonstrated the incorporation

FEDERATION PROCEEDINGS

Volume 16

of amino acids into freshly prepared rat liver Me
in the presence of an ATP-generating system and
supernatant solution (S) obtained by centrifuga-
tion at 105,000 g. C4-glutamic acid was utilized
rapidly in the Mc-S system with most of the total
incorporated radioactivity in the TCA insoluble
material of S. However, over 90% of the counts
could be removed from the S proteins by treat-
ment with thioglycolate or dialysis against
cysteine. In order to facilitate purification and
obtain a preparation free of thioesterases and the
enzyme system forming y-glutamylcysteine, the
requirements for amino acid incorporation were
studied with the aid of C'-leucine and C-
phenylalanine. A stable Mc preparation was
obtained by lyophilization of a 15,000 X g super-
natant fluid of rat liver homogenate. Amino acid
incorporation into the Mc fraction of this prepara-
tion required S protein, and an ATP-generating
system. The incorporation was stimulated several
fold by neutral salt and potassium ions. High
concentrations of ATP, AMP and polyvalent
anions were inhibitory. For comparative purposes
the requirements for incorporation into acetone-
dried Me were also studied. The stability of the
lyophilized Mc preparation has greatly facilitated
the study of the detailed requirements and kinetics
of amino acid incorporation.

1127. Formation of hydroxyaspartic acid from
dihydroxyfumaric acid and glutamic acid.
H. J. Sauiacn (introduced by R. W.
McGitvEry). Dept. of Physiological Chemistry,
Univ. of Wisconsin Med. School, Madison.

In the course of studies dealing with the mecha-
nist of serine synthesis it was observed that a
system consisting of dihydroxyfumaric acid,
glutamic acid and various tissue preparations gave
rise to an unknown ninhydrin-reacting substance.
The unknown compound was retained on an
Amberlite IR-4B column, acetate form at px 5,
and was cleaved by periodate with the liberation
of ammonia. The compound had the same Rr as
synthetic hydroxyaspartic acid in a variety of
solvents. These results indicate that the unknown
compound is hydroxyaspartic acid formed by
transamination between oxaloglycolic acid (the
keto form of dihydroxyfumaric acid) and glutamic
acid. The enzyme is present in heart, liver, kidney
and brain obtained from a number of sources and
has been partially purified from sheep brain.
Dialysis of the enzyme or the presence of EDTA
(Versene) in the reaction mixture resulted in a
marked reduction in hydroxyaspartate formation,
but activity could be restored by the addition of
Mg** ions. In the reverse reaction, hydroxy-
aspartate and a-ketoglutarate transaminated
with the formation of glutamate as one of the
expected products. It was identified by paper

—_— * <= were ey OH

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March 1956

chromatography and with a specific glutamic acid
decarboxylase from E. coli. There was no decrease
in activity upon dialysis nor EDTA inhibition
when the reaction was approached from this
direction. (Supported in part by the Public
Health Service.)

1128. Mechanism of iron accumulation in
liver slices. Paut Satrman, Harry FRiscu,
RoserT FIsKIN AND THEODORE ALEX (intro-
duced by ARNOLD WaRE). Dept. of Biochemistry
and Nutrition, Univ. of Southern California, Los
Angeles.

By studying the effects of various physical and
chemical environments on the accumulation of
iron by rat liver slices, we have demonstrated that
metabolic energy is not directly coupled to ion
accumulation. It appears that the iron is sorbed
by a specific iron binding entity within the cell,
possibly ferritin. Kinetic studies of the process
exhibit 2 important characteristics: 1) the rate of
accumulation is enhanced at higher temperatures,
2) the capacity of the iron binding entity is
increased at higher temperatures. We postulate a
2-step process to explain the results: /) a diffusion
controlled transport to the sites on the iron
binding entity, 2) subsequent binding of the iron
by a reaction or series of reactions whose rate is
rapid compared to the rate of transport. Kinetic
experiments using rat liver cell suspensions show
the same characteristics as the experiments using
slices. However, when the tissue is homogenized,
the uptake of iron is practically instantaneous.
We conclude that the iron binding entity must be
in direct contact with the cell membrane, and that
the rate limiting diffusion reaction is the transport
of the ion across the membrane barrier. Any
alteration either in the amount of iron binding
entity or in its binding capacity could account for
the storage of iron observed in various patho-
logical conditions.

1129. Cysteamine oxidase. RicHarp SALVADOR*
AND RoscoE QO. Brapy. Natl. Inst. of Neuro-
logical Diseases and Blindness, Bethesda, Md.,
and Dept. of Pharmacology, George Washington
Univ. School of Medicine, Washington, D.C.

We have recently observed the presence of an
enzyme in soluble extracts of pigeon liver acetone
powder which catalyzes the oxidation of 2-mer-
captoethylamine when incubated in the presence
of 2,3,5-triphenyltetrazolium chloride. The en-
zyme has been purified 15-fold by fractionation
with ammonium sulfate (0-30% saturation),
alcohol (0-21% ethanol), and elution from calcium
phosphate gel. Diphosphopyridine nucleotide is
required although triphosphopyridine nucleotide
shows slight activity in the crude extract.
N-acetyl-2-mercaptoethylamine is oxidized about
jj as rapidly as cysteamine, whereas pantetheine,

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS 345

mercaptoethanol, cysteine, glutathione, and
cystamine are inactive as substrates for this
enzyme. Coenzyme A appears to be oxidized only
very slightly in the partially purified system.
When S*-labeled cysteamine was incubated with
the enzyme precipitating between 0-21% ethanol,
which still exhibited diaphorase activity, one of
the major products appeared to be cystamine
disulfoxide. Cell-free extracts of C. kluyveri
contain a similar enzyme although the product of
this reaction has not been identified. This prepara-
tion differs somewhat from that obtained from
pigeon liver since 2-mercaptoethanol as well as
cysteamine was rapidly oxidized by the enzyme
system obtained from C. kluyvert.

1130. Induced formation of SIII and SVIII
pneumococcal’ polysaccharide depoly-
merases by B. palustris. T. Sautzman, F.
DELAFIELD AND A. M. TorrRiAni (introduced
by A. M. PaprpENHEIMER, JR.). Dept. of Micro-
biology, New York Univ. College of Medicine,
New York City.

B. palustris is capable of forming two distinct
and specifically inducible enzymes, SIII- and
SVIII-depolymerases, which hydrolyse the corre-
sponding pneumococcal polysaccharides. In-
duction and enzyme formation in growing cultures
have been studied using a sensitive viscosometric
method to estimate enzyme activity. Good growth
of organisms occurs on a simple medium con-
taining salts, glycine, glutamate, biotin, PABA,
nicotinamide and thiamine plus an energy source.
When succinate is used as chief source of carbon,
depolymerase formation is induced by small
amounts of corresponding polysaccharide. In the
presence of glucose or maltose, however, a diauxic
type of growth curve results and no enzyme is
formed until the inhibiting sugar has been com-
pletely utilized. Cellobiuronic acid, the aldo-
biuronic acid obtained by acid hydrolysis of SIII,
fails to induce enzyme formation by itself, but
when used as chief carbon source it does not
inhibit induction of SIII depolymerase by SIII.

1131. Pyrrole-containing precursor of pro-
digiosin. Ursuta V. SAnTER AND HEnry J.
VoGEL (introduced by Davin M. Bonner).
Dept. of Microbiology, Yale Univ., New Haven,
Conn.

Of two Serratia marcescens mutants unable to
synthesize the tripyrrylmethene pigment pro-
digiosin, one (9-3-3) was found to excrete a sub-
stance that allowed the other (W-1) to produce
this pigment. The substance excreted has been
isolated in pure crystalline form and, by isotope
techniques, demonstrated to be an actual pre-
cursor of prodigiosin. Elementary analysis agrees
with the formula CiH»O2Ne, including one
methoxyl group. The precursor does not melt at

346 FEDERATION PROCEEDINGS

250°; it is neutral, colorless, and in ethanolic
solution absorbs strongly in the ultraviolet, with
maxima at 363 and 254 mu. Its infrared spectrum
gives no indication of ester, lactone, or free
hydroxyl] or carbonyl functions; an amide group
is not ruled out. The crystalline compound gives a
test for pyrrole with p-dimethylaminobenzalde-
hyde. Reduction of the precursor with hydrogen
and platinum oxide, followed by oxidation with
permanganate, leads to the formation of proline,
detected by bioassay. The pyrrole ring of the pre-
cursor probably is incorporated as such into
prodigiosin. Another portion of the precursor may
give rise to a second pyrrole ring, but presumably
not the amylmethylpyrrole, of prodigiosin. The
amyl-containing moiety may arise by a separate
biosynthetic sequence. A bifurcated pathway of
prodigiosin synthesis is indeed indicated by the
additional finding that strain W-1 excretes an
unidentified substance that permits prodigiosin
formation in strain 9-3-3; moreover, pigment-
mutant P-18 excretes two substances to which
strain 9-3-3 and W-1 can respectively respond with
pigment formation.

1132. Metabolism of folic acid by human
subjects. Lucia S. Santiaco,* M. ANN
Wa.porr,* MartHa McMILuan* anp HELEN
T. Parsons. Univ. of Wisconsin, Madison.

In conformity with a previous report from this
laboratory, a shift was noted in the ratio of
citrovorum factor to total folic acid activity (for
S. faecalis) in the urines of human subjects over a
4-day period on 1 mg PGA dosage. A 1:3 ratio
(before dosage) shifted to an average 1:40 ratio
by the 2nd day of dosage when CF had reached
its peak output, and to 1:90 by the 4th day when
CF output had declined for 2 days. Assays were
made immediately on completion of 24-hr. urinary
collections. Storage of the urinary samples for
24 hr. at 6° resulted in loss in CF values but, in
many in¢fances, significantly higher values for
total FA; some FA values were 50% higher at the
end of 24 hr. storage. No clear evidence that bac-
terial action was chiefly responsible for the
changes on storage was procured by autoclaving
the urines or by seeding them with S. faecalis
before storage.

1133. Microbiological studies on aspartic acid
metabolism. H. E. SAUBERLICH AND GORDON
Gurorr.* Dept. of Animal Husbandry and
Nutrition, Alabama Polytechnic Inst., Auburn.
The requirements of lactic acid producing

bacteria for aspartic acid or asparagine were

studied. As a result of these studies, a procedure
was developed whereby both asparagine and
aspartic acid could be measured microbiologically
with a strain of Lactobacillus delbriickii-5. High

Volume 16

amounts of glutamic acid in the medium com-
pletely inhibited the growth of the organism when
cultured in the presence of aspartic acid. No
inhibition occurred when asparagine was used.
The method was employed on natural materials
and in a study on the disappearance of injected
aspartic acid and asparagine from the blood of
rats. Other experiments were devised to study the
metabolic functions of aspartic acid. When special
minimal media were used, aspartic acid was
required by bacteria that normally did not require
it. For example, L. arabinosus-17-5 did not require
purines, pyrimidines or threonine when adequate
p-aminobenzoic acid and pyridoxal were in the
media, but when purines, pyrimidines and threo-
nine were omitted from the medium, L-aspartic
acid was essential. The p-form was inactive. If
the purines and pyrimidines were omitted, then
either threonine or aspartic acid was necessary.
In the absence of vitamin Bs, lysine and alanine
were also required in addition to aspartic acid,
threonine, purines and pyrimidines. Thymidine
and a purine (hypoxanthine) were essential in
the absence of p-aminobenzoic acid. Ureido-
succinic acid was active. Tracer studies with
C14-labeled aspartic acid revealed incorporation of
activity into purines, pyrimidines, threonine,
serine, glycine, lysine, histidine, diaminopimelic
acid and arginine. The incorporation depended
upon the position labeled in the aspartic acid.
(Supported in part by Atomic Energy Com-
mission contract No. AT-(40-1)-1674.)

1134. Steroid 118-hydroxylation by a human
testicular tumor. KENNETH SAVARD, RALPH
I. Dorrman, Bruty Baacetrt* anp Lewis L.
ENGEL. Worcester Fndn. for Exptl. Biology,
Shrewsbury, Mass., and Huntington Memorial
Hosp. of Harvard Univ. at Massachusetts General
Hosp., and Dept. of Biological Chemistry, Harvard
Med. School, Boston.

Tissue slices, obtained from an interstitial-cell
tumor of a testis removed from a 5}-year-old
boy, were incubated with testosterone-4-C™ in
human serum containing added fumarate, glucose,
equine and chorionic gonadotrophins and pitui-
tary FSH, for 3 hr. at 37° under 95% O2-5% CO».
Carrier steroids were added to the incubation
mixture and it was extracted by the Folch-Pi
method. The extract was freed of nonpolar lipide
by partitioning between ligroin and 90% methanol.
The resulting polar material on conventional
treatment gave a neutral ketonic fraction with
which the major proportion of the radioactivity
was associated. The individual steroids were
isolated from this fraction by paper chroma-
tography in the toluene-propylene glycol and
ligroin-propylene glycol systems. Radioactive
118-hydroxytestosterone, 118-hydroxy-4-andros-

> 2 ~~ OP @ =. 2 tt & © @ sh eo @ @ bet oes es A 2 oe ek

Cc as

ve 16

com-
vhen
No
ised.
rials
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ecial
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nine
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idine
al in
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with
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melic
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Com-

man
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ology,
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1-cell
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ation
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were
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March 1956

tene-3 , 17-dione, andrenosterone and 4-androstene-
3,17-dione were obtained. The conversion of
testosterone to the above individual products was
in the range of 0.1-1.0%. The following types of
procedures were employed consecutively to
establish the radiochemical purity of each isolated
steroid: chromatography followed by crystalliza-
tion from several solvent systems, chemical
conversion to a derivative followed by chroma-
tography and crystallization.

1135. Phosphopeptides from chymotrypsin
and trypsin after inactivation by P* labeled
DFP and Sarin. Norwoop K. ScHAFFEr,
Rosert R. EnGiE,* LEon Stmet,* Ricwarp W.
Drisko* AND SrpNEY HarsHMan.* Enzyme
Chemistry Branch, Chemical Corps Med. Labs.,
Army Chemical Center, Md.

The inactivating reaction of diisopropyl phos-
phorofluoridate (DFP) with chymotrypsin and
trypsin results in the formation of monophos-
phorylated derivatives of these enzymes. The
configuration about the DFP-binding site of
chymotrypsin previously reported (Federation
Proc. 14: 275, 1955) has been extended through
the isolation of glycylaspartylphosphoserylglycine
from a 12 n HCl hydrolysate of the P*-labeled
DFP derivative. The presence of an aspartyl,
rather than an asparaginyl, residue has been
established by means of Edman’s phenylisothio-
cyanate method. Isopropyl methylphosphono-
fluoridate (Sarin) also inactivates chymotrypsin
and trypsin forming monophosphorylated deriva-
tives. Hydrolysis of the P*?-labeled Sarin deriva-
tive of chymotrypsin (12 n HCl, 37°, 3 days) and
Dowex 50 chromatography has yielded, besides
the corresponding previously found di- and tri-
peptides, a peptide containing glycine, aspartic
acid, methylphosphonylserine, glutamic acid,
alanine and valine. Its N-terminal sequence
(Edman’s method) appears to be glycine-aspartic
acid and carboxypeptidase liberates both alanine
and valine. A papain digest of Sarin-chymo-
trypsin has yielded a peptide containing histidine,
proline, leucine, cystine and threonine in addition
to the 6 residues found in the peptide from HCl
hydrolysis. Chymotryptic digestion of this peptide
results in the loss of histidine and proline. Hy-
drolysis of Sarin-trypsin with 12 n HCl and
chromatography has yielded the following phos-
phopeptides: aspartylmethylphosphonylserine,
methylphosphonylserylglycine, and aspartyl-
methylphosphonylserylglycine. Hence, the resi-
dues adjacent to the alkyl-phosphate binding
serine residue in both chymotrypsin and trypsin
are identical.

1136. Acid hexokinase from yeast. THOMAS
ScHARFF AND ASER ROTHSTEIN (introduced by

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS 347

H. C. Honege). Div. of Pharmacology, Dept. of
Radiation Biology, Atomic Energy Project, Univ.
of Rochester, Rochester, N. Y.

An acetone powder extract of baker’s yeast
contains 2 hexokinases: the well-known enzyme
with a pH optimum of about 8.4, and another
which has a pH optimum of 5-6. Activity peaks
were found in 2 pH regions with the extracts, but
only in the pu 8.4 region with purified hexokinase.
The ratio of activity at pH 5.5 to that at 8.4 was
used as an index of the relative amounts of the 2
enzymes. Ratios of 0.23 and approximately 0.5
were found for purified hexokinase and for the
extracts respectively. The ratio in the extracts
was varied in several ways: lowered ratios were
found in acetone precipitates. Blood serum from a
rabbit immunized against the purified hexokinase
effected a rise in ratio from an initial value of
0.48-0.67, the increase resulting from a loss of
activity in the px 8.4 region. In a starch electro-
phoresis experiment conducted at pu 6.5 the
negative side of the starch block yielded an active
fraction with a ratio of 1.9, showing a marked
purification of the acid enzyme. The isoelectric
point of the acid hexokinase is above 6.5, in
contrast to the reported isoelectric point of 4.8
for crystalline hexokinase. (This paper is based on
work performed under contract with the Atomic
Energy Commission at the Univ. of Rochester
Atomic Energy Project, Rochester, N. Y.)

1137. Formation and binding of histamine in
vitro. RicHarp W. ScuayEr. Rheumatic Fever
Research Inst., Chicago, Ill.

This laboratory has reported that rat abdominal
skin incubated with C" L-histidine forms a small
amount of C'* histamine and binds it in stable
condition. The amount of histamine bound is
increased by prior adrenalectomy and decreased
by pretreatment with cortisone (Am. J. Physiol.
182: 54, 1955). Of the other tissues tested, by far
the most active in formation and binding of
histamine is the pyloric portion of the stomach.
Unlike most tissues, in vitro histamine binding by
stomach is increased by cortisone treatment and
decreased by adrenalectomy. Rat lung appears to
be the most useful tissue for evaluating ability of
cortisone analogs to suppress histamine formation.
Pretreatment of rats with 1 mg prednisone per
day for 3 days resulted in 61% loss of activity of
lung tissue. With 5 mg per day the loss was 84%.
Recently we have found that the free mast cells
of rat peritoneal fluid have a pronounced ability
to decarboxylate t-histidine and to bind the
resulting histamine. Much of the histamine of the
body is believed to reside in mast cells. Hence, an
uncomplicated system is available for studying
formation and binding of histamine by mast cells.
A soluble histidine decarboxylase can be obtained

348

from these mast cells and its properties are being
investigated. The buffy coat of rabbit blood is also
very active in formation and binding of C
histamine. Rabbit platelets, unlike those of other
species, are high in histamine and are probably
the active element. A soluble histidine decarboxy]-
ase can also be obtained from the rabbit buffy
coat.

1138. Hydrodynamic properties, molecular
configuration and reactivity of bovine
serum albumin. H. A. Scueraca, G. I. Lozs*
AND M. L. Waaner.* Dept. of Chemistry, Cornell
Univ., Ithaca, N. Y.

The reactivity of bovine serum albumin (BSA)
towards low molecular weight molecules and ions
exhibits anomalies at low and high pu. In order to
ascertain the contribution of the molecular con-
figuration to the reactivity, the hydrodynamic
properties of BSA were investigated at pH 4.0
and 5.13. Viscosity, sedimentation and diffusion
measurements were made in 0.5 m KCl to provide
the independent hydrodynamic quantities re-
quired for the determination of the configuration.
A molecular weight of 67,400 was obtained from
the sedimentation and diffusion coefficients. The
value of the computed hydrodynamic parameter 6
indicates that the hydrodynamic properties of
BSA can be interpreted in terms of an equivalent
sphere at both px 4.0 and 5.13, the volume at px
4.0 being 10% greater than that at pH 5.13. This
increase in volume at the lower pH can account
for part of the anomalous reactivity insofar as it
influences the electrostatic contribution to the free
energy. The remainder of the anomaly might be
attributed to a variation in the observed px of the
groups involved due to the reversible formation
and breakage of internal hydrogen bonds between
the side chain polar R groups of the protein
(Laskowski, M., Jr. anp H. A. ScHmRAGA.
J. Am. Chem. Soc. 76: 6305, 1954). The changes in
volume (and concomitant penetration of solvent
into the molecular domain) accompany the forma-
tion and breakage of the hydrogen bonds.

1139. Isolation of chondroitinsulfuric acid
from normal human plasma. Sara ScHILLER
AND Katuryn F. Dewey.* LaRabida Jackson
Park Sanitarium and Dept. of Pediatrics, Univ.
of Chicago, Chicago, IIl.

Two liters of plasma from normal humans was
digested with a mixture of proteolytic enzymes
and precipitated with trichloroacetic acid. The
trichloroacetic acid-soluble material was dialyzed
and concentrated. Addition of alcohol in the
presence of sodium acetate caused the precipita-
tion of 60 mg of a mixture of crude polysac-
charides. Electrophoresis of an aliquot on paper
demonstrated the presence of 2 spots, one having

FEDERATION PROCEEDINGS

Volume 15

the same mobility and staining characteristics
with toluidine blue as hyaluronic acid (HA) while
the other behaved like chondroitinsulfuric acid
(CSA). The remainder of the material was sub-
jected to zone electrophoresis on celite. Uronic
acid was found in eluates of fractions with the
same mobility as CSA. Chemical analysis of the
combined eluates revealed the presence of sulfate
and equimolecular concentrations of hexosamine
and uronic acid, as determined by the carbazole
reaction. The material was completely hydrolyzed
by testicular hyaluronidase as determined by the
reduction in turbidity. In view of the action of the
testicular enzyme and the degree of color develop-
ment with the carbazole reagent, the CSA in
plasma appears to be identical with that of carti-
lage. (Aided by a grant from the Natl. Heart Inst.,
Natl. Insts. of Health, PHS.)

1140. Metabolism of thetins in yeast (Toru-
lopsis utilis). F. ScH~tenk anp R. E,
DerPatma.* Argonne Natl. Lab., Lemont, Ill.

The ability of several thetins toform methionine
from homocysteine by transmethylation has been
tested with yeast (Torulopsis utilis). Dimethyl-
acetothetin and dimethylpropiothetin failed to
give significant amounts of methionine, while
S-methylmethionine reacted readily (cf. S. K.
Suaprro. Biochim. et Biophys. Acta 18: 134, 1955)
according to the equation: S-methylmethionine +
homocysteine — 2 methionine. In yeast, betaine
and choline were found to be inactive as substi-
tutes for S-methylmethionine. Attempts were
made to study the reaction : S-adenosylmethionine
+ homocysteine — S-adenosylhomocysteine +
methionine. This was observed only in living
yeast cells and the rate was slow; S-adenosyl-
methionine apparently is less effective than
S-methylmethionine as a methyl donor in Toru-
lopsis cells with homocysteine as the acceptor.
This is surprising because yeast has an unusual
ability to produce and store S-adenosylmethio-
nine, if a suitable organic sulfur supplement is
provided in the culture medium; as much as 40-60
uM/gm of dry cells has been obtained routinely.
(Work performed under the auspices of the Atomic
Energy Commission.)

1141. Ribonucleic acid and protein balance in
phosphate- and sulfate-starved bakers’
yeast. GERHARD Scumipt, Krikxor SErRaI-
DARIAN* AND 8.J. THANNHAUSER. Dept. of Bio-
chemistry and Boston Dispensary, Tufts Univ.
Med. School, Boston, Mass.

We reported in earlier papers that bakers’ yeast
incubated for 24 hr. in a medium deprived of
phosphate, but containing ammonium sulfate,
glucose and potassium salts approximately
doubles its protein content (determined by dif-
ference between total N and N in the boiled

act
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pre
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was
or |
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onic

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zed

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een
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rine
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March 1956

extract), while its total purines decrease during
this period by 5-8%. The RNA content decreases
during this period by 30-40%, whereas the acid-
soluble purine compounds double their amounts
at the expense of RNA. This suggests the working
hypothesis of intracellular utilization of RNA for
the formation of essential acid-soluble nucleotides,
in line with the concept presented by M. Soodak
at the Gordon Conference on Proteins and Nucleic
Acids, 1593. After transfer of the cells to medium
supplemented with phosphate, RNA increased
within 4 hr. by 200%, whereas the protein-N
increased by only 30%. Several observations
suggest the interpretation that RNA synthesis is
dependent on preceding or simultaneous protein
formation. 1) No RNA is formed when protein
synthesis is suppressed a) by omitting sulfate
during the incubation in P-deprived and in
P-containing medium or b) by incubating yeast
which had been starved of phosphate and sulfate
in a P-supplemented medium containing ethionine.
2) Even when protein formation is largely sup-
pressed by ethionine, the yeast cells double their
RNA contents if sulfate was present during
P-starvation, enabling the yeast to accumulate
protein prior to their transfer to the medium
containing ethionine and phosphate.

1142. Deoxyribosidic compounds in liver and
hepatoma. WALTER C. SCHNEIDER AND LEONA
W. BrownELu.* Natl. Cancer Inst., Natl. Insts.
of Health, PHS, Bethesda, Md.

The deoxyribosidic compounds, present in acid
soluble extracts of tissues and presumably repre-
senting precursors of deoxyribonucleic acid, have
been studied in normal and regenerating rat liver
and in the Novikoff hepatoma. The total amount
of these compounds, as measured microbio-
logically, was about the same in the tumor and in
normal liver (10 ug/gm) but was increased over
50% in the liver after partial hepatectomy before
actual liver regeneration occurred (24 hr. post-
operative). The type of deoxyribosidic compound
present in these tissues was, however, considerably
different. Using improved methods of isolation, it
was found that deoxycytidine accounted for 90%
or more of the normal liver deoxyribosidic com-
pounds, while in the tumor 50% of the deoxy-
ribosidic compounds were isolated as the nucleo-
sides deoxycytidine, deoxyuridine and thymidine
in the approximate ratio 5:1:1. The remainder of
the hepatoma deoxyribosidic compounds were
precipitable with barium acetate and ethanol and
were strongly retained on Dowex-1-formate
columns, suggesting that they were deoxynucleo-
tides or polynucleotides. Since 25-30% of the
deoxyribosidic compounds of the liver 24 hr. after
partial hepatectomy was also found to have
nucleotide-like properties, these compounds would

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS 349

seem to be important in growth. The isolation and
identification of the nucleotide-like compounds is
in progress.

1143. Effect of Triton WR-1339 on lipoprotein
lipase activity of rat plasma. Micuar. C.
Scuotz anp A. Scanu (introduced by E. Haas).
Research Div., Cleveland Clinic Fndn. and Frank
E. Bunts Educational Inst., Cleveland, Ohio.
Surface active agents inhibit various enzyme

systems. Since lipoprotein lipase (‘clearing
factor’) may play a role in regulating fat trans-
port, its inactivation by Triton might explain
hyperlipemia which results from Triton injection.
In one, in vivo, series of experiments 50 mg of
Triton was administered intravenously into 10
rats, 25 min. later heparin (5 u/100 gm) was
injected into the heart and 5 min. later the blood
withdrawn from the heart. The plasma was
obtained by centrifugation at 0°C and stored
frozen. Lipoprotein lipase was measured a) by
incubation of postheparin plasma with cocorut oil
substrate and measuring both the fall in optical
density of the mixture at 660 my and b) from the
production of free fatty acids. All determinations
were carried out in duplicate or triplicate. Both
procedures showed that lipoprotein lipase activity
of the plasma of Triton-treated rats was almost
completely inhibited in comparison with the
control animals. In the second, in vitro, series of
experiments, 10 rats were used. Plasma was
obtained 30 min. after the animals had been
injected intravenously with 50 mg. of Triton. This
plasma, when added to a system containing post-
heparin plasma plus coconut oil substrate, totally
inhibited enzymatic clearing activity. Hence, by
in vivo and in vitro experiments, it was demon-
strated that Triton inhibits lipoprotein lipase
activity. Inhibition of lipoprotein lipase may
therefore be a mechanism by which injected Triton
causes hyperlipemia.

1144. Utilization of glycine, succinate and 6-
aminolevulinic acid for heme synthesis.
Martin P. ScouLMAN AND Dan A. RICHERT.
Dept. of Biochemistry, State Univ. of New York
Med. College, Syracuse.

Heme was synthesized in red cell suspensions
and hemolysates of vitamin Be-deficient ducklings
from glycine-2-C-14 and succinate-2-C-14 at less
than half the rate found with control duck cells.
Addition of pyridoxal-5-phosphate in vitro stimu-
lated the incorporation of the labeled compounds
into heme in Be-deficient red cell preparations
(J. Am. Chem. Soc. 77: 6402, 1955). Pyridoxal,
pyridoxamine and pyridoxine were ineffective.
Heme synthesis from 6-aminolevulinic acid-2,3-
C-14 proceeded at the same rate in Be-deficient
and normal ducklings and was not stimulated by

350 FEDERATION PROCEEDINGS

the addition of pyridoxal-5-PO,. Pyridoxal-5-PO,
did not stimulate heme synthesis from glycine-2-
C-14 when Beg-deficient duck cells were previously
washed at least 5 times with saline. Plasma re-
stored the pyridoxal-5-PO, effect to the washed,
deficient cells and the same degree of stimulation
by pyridoxal-5-PO;, was obtained by adding
FeCl; to a saline suspension of washed deficient
cells instead of the plasma. Red cells and hemol-
ysates from control ducklings incorporate labeled
glycine and succinate into heme several times
better than cells from pantothenic acid-deficient
ducklings; the utilization of 6-aminolevulinic
acid for heme synthesis was unaffected by the
pantothenic acid deficiency. Addition of coen-
zyme A in vitro to the deficient cells did not
restore heme synthesis from glycine to succinate.
It can be concluded that both pyridoxine and
pantothenic acid deficiencies exerted their effects
on heme synthesis in the utilization of glycine
and succinate for the formation of 6-aminolevu-
linic acid. (Aided by Grant C-1852-C2 from Natl.
Insts. of Health, PHS.)

1145. Beta-globulins of sera with altered
electrophoretic patterns. JuLIUS ScHULTz,
GrorcE F. Grannis,* Haze, B. KimMEx* anp
Harry Suay. Fels Research Inst., Temple Univ.
School of Medicine, Philadelphia, Pa.

In the normal rat, dog and man the average
ratio of alanine to threonine in the beta-globulins
is 1.1-1.2, and in the gamma-globulins the ratio
in all 3 species is 0.8, when these proteins are
prepared from sera by zone electrophoresis on
starch (Arch. Biochem. & Biophys. 57: 174, 1955).
In the present series these values were compared
with those obtained in sera where the beta-
globulins are found to be split into beta-1 and
beta-2 globulins. Thus: for beta-1, beta-2 and
gamma-globulins, respectively; 1- and 2-day
fasted rats, 1.2, 0.75, 0.65; 7-day fasted rats, 0.9,
0.7, 0.45; precancerous rats, 1.3, 1.1, 0.7; cancerous
rats, 1.3, 1.2, 0.8; multiple myeloma of man, beta-
myeloma, 1.0; gamma-myeloma, 0.7; beta-globu-
lin of the latter, 1.3. The same ratios calculated
from the 4 myeloma proteins reported by Smith
et al. (J. Biol. Chem. 216: 601, 1955), 0.8-1.0.
These data suggest that the extra beta-globulins
of the cancerous and precancerous rat may be of
hepatic origin, while in the fasted rat the extra
beta-globulin is characteristic of proteins of the
RE system similar to the gamma-globulins and
the myeloma proteins.

1146. Enzymatic degradation of deoxyribonu-
cleic acid. VERNE N. ScHUMAKER,* GLEN E.
Ricuarps* anp Howarp K. ScHacuMan. Virus
Lab., Univ. of California, Berkeley.

In a recent paper from this laboratory dealing

Volume 16

with structural models for deoxyribonucleic acid
(DNA) with particular emphasis on the two-
strand model proposed recently by Watson and
Crick it was pointed out that a detailed study of
the kinetics of the enzymatic digestion of DNA
might yield information about different models.
For example, a one-strand structure would be
split at each attack by the enzyme. A two-strand
structure would require at least two attacks and
therefore the efficiency of splitting would increase
with the square of the number of attacks. A two-
strand structure with some ‘preformed gaps,’ a
model proposed in the above-mentioned paper,
would degrade in a manner intermediate between
a single-strand model and a continuous two-
strand model. This communication presents new
data on the viscosity and sedimentation changes
as a function of the number of enzymatic attacks
on the DNA macromolecule. The number of en-
zymatic attacks is determined in a ‘pu-stat’ by
measuring hydrogen ion liberation resulting from
the cleavage of the phosphodiester bonds. Simple
equations, obtained from a detailed statistical
theory for the degradation of macromolecules,
are presented, and these equations are used with
the experimental data in a discussion of different
models for DNA. The results of this approach
indicate that DNA is essentially a continuous
two-strand structure, having at most one ‘pre-
formed gap’ for every 3000 nucleotides.

1147. Incorporation of radioactive lysine into
protein. RicHARD ScHWEET (introduced by
Norman Horowi17z). Calif. Inst. of Technology,
Pasadena.

Radioactive lysine is incorporated into the pro-
teins of a soluble enzyme fraction obtained from
guinea pig liver. Because of the unusual features
of the incorporation process (Federation Proc. 14:
277, 1955), the mode of linkage of incorporated
lysine has been studied in detail. After treatment
of the radioactive protein with dinitrofluoro-
benzene or nitrous acid, followed by hydrolysis,
all of the radioactivity was found in a-DNP-
lysine and a-hydroxy-e-aminocaproic acid, re-
spectively. Thus, incorporated lysine is linked by
its e-amino group. This type of bond has not been
previously found in protein. To determine the
specific group to which the lysine was bound,
partial hydrolysates of radioactive protein were
made. The protein was digested with pepsin,
trypsin and chymotrypsin, oxidized with per-
formic acid and then hydrolyzed further with
concentrated hydrochloric acid for 5-7 days.
Little free radioactive lysine was produced.
Radioactive peptides were purified by chromatog-
raphy on Dowex-50 and on paper and by other
techniques. Two radioactive peptides have been
isolated so far. In addition to lysine, both of

eq
cr’
ca

col

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tiv.
by

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114
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li
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een

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March 1956

these contain glutamic acid and unidentified
components. Further work on the structure of
these compounds will be required to prove that
the lysine is actually linked to glutamic acid,
although this seems probable.

1148. Mechanism of action of heart muscle
lactic dehydrogenase. GEoRGE W. ScHWERT
AND Yasuo TaKENAKA.* Dept. of Biochemistry,
Duke Univ. School of Medicine, Durham, N. C.
It has been shown previously from kinetic and

equilibrium data that the mode of action of the
crystalline lactic dehydrogenase of heart muscle
can be described by two alternate mechanisms.
In one of these, the enzyme is assumed to form a
complex with either substrate or coenzyme and
then to form a ternary complex with the remain-
ing reactant. In the other case, a compulsory
pathway is postulated in which the enzyme must
form a complex with one reactant before it can
combine with the second reactant (Hakata,
GuAIp AND Scuwert. J. Biol. Chem. In press).
Direct binding measurements, made by the ultra-
centrifugal separation technique, indicate that
DPN is bound at approximately 4 sites on the
enzyme molecule and that the dissociation con-
stant of the enzyme—DPN complex is of the order
of 3 X 10-* m at 25°, px 6.80. Pyruvate, on the
other hand, is not bound to a measurable extent.
It has also been found that there are 4 sulfhydryl
groups per mole of enzyme which react rapidly
with p-chloromercuribenzoate and that inac-
tivation of the enzyme by this reagent is inhibited
by DPNH but not by pyruvate. These results,
taken together with the spectroscopic demonstra-
tion of an enzyme—DPNH complex by Chance
and Neilands (J. Biol. Chem. 199: 384, 1952) in-
dicate that the reaction follows a compulsory
pathway in which the enzyme must combine with
coenzyme before substrate can be bound.

1149. Phytic and other acids of the potato
tuber. Sigmunp ScowimMER. Western Utiliza-
tion Research Branch, Agric. Research Service,
U.S. Dept. of Agriculture, Albany, Calif.
Previous studies (ScHWIMMER et al. Agr. Food

Chem. 3: 257, 1955) demonstrated the presence in

acid extracts of potatoes of nonpolysaccharide

material, precipitating at pH 8.2 in aqueous or
ethanolic solutions, which interfered with sub-
sequent procedures for phosphate ester separa-
tion. This fraction, after purification by repeated
solution in cold 0.2 N HCl and precipitation at
pH 8.2 with ammonia, has been identified by
analytical, spectrographic and chromatographic
methods as a mixture of the dicalcium, monomag-
nesium salts of inositol hexa- and penta- phos-
phoric acids, constituting about 20-25% of the
total tuber phosphorus. Purification of the crude
precipitate removed about 5-10% of impurities

eee tae

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS 351

which were identified chromatographically as
malic, citric and aconitic acids as well as poly-
mers of glucose, fructose, pentose and uronic
acid. Paper chromatography with 3 different
solvent mixtures (SCHWIMMER et al. Agr. Food
Chem. 3: 257, 1955; Stark et al. Anal. Chem. 23:
413, 1951; ANDERSON. Nature 175: 863, 1955) and
paper electrophoresis revealed 2 adjacent spots
for both the purified potato and commercial
phytate cation-treated preparations.

1150. Pyrophosphatases of swine brain. U.
S. Sean (introduced by F. BinkuEy). Div. of
Basic Health Sciences, Emory Univ., Emory
University, Ga.

Two enzymes responsible for the hydrolysis of
inorganic pyrophosphate have been prepared
from swine brain. Both have been solubilized by
treatment of the 900 X g supernatant of an aque-
ous homogenate with digitonin, octanol and
chloroform. They have been purified several
hundredfold with a nearly quantitative yield.
One hydrolyzes pyrophosphate and ATP and is in
other respects similar to the enzyme reported by
Olson and Binkley. The other enzyme hydrolyzes
only inorganic pyrophosphate and is similar in its
behavior to the inorganic pyrophosphatase of
erythrocytes. No more than 3% of this latter
activity can be attributed to residual blood in
the tissue. When fully activated, the inorganic
pyrophosphatase has an activity about one order
of magnitude greater than that of acetylcholines-
terase on the basis of moles of substrate split per
gram of fresh tissue per hour. This order of ac-
tivity depends upon optimal concentrations of
magnesium ion and glutathione. It has been
demonstrated that the activation with metal ion
depends on stage of purification, concentration of
substrate, buffer, ionic strength, pH and adventi-
tious inhibitors and activators.

1151. Interrelationships between prothrom-
bin and certain derivatives of prothrombin.
Wa.ttrer H. Seecers, Surrtey A. JoHNSON*
AND Norma ALKJAERSIG.* Dept. of Physiology
and Pharmacology, Wayne Univ. College of
Medicine, Detroit, Mich.

Purified bovine prothrombin preparations can
be converted to autoprothrombin I with calcium
ions, a small amount of Ac-globulin, and purified
platelet factor 3. The reaction can be reversed to
a limited degree with liver mitochondria. Auto-
prothrombin I catalyses the slow activation of
purified prothrombin with SPCA plasma. Auto-
prothrombin I and prothrombin can both be
converted to thrombin in 25% sodium citrate
solution; however, an intermediate forms which is
called citrate autoprothrombin. Like autopro-
thrombin I it is an accelerator in the activation
of purified prothrombin to thrombin when throm-

352

boplastin, calcium ions and Ac-globulin are used.
Another derivative of prothrombin forms when
suitable amounts of purified thrombin and Ac-
globulin are added to prothrombin preparations.
This derivative is called autoprothrombin II,
its concentration drops to zero in Dicumarol
plasma, and we believe it is platelet cofactor II,
Christmas factor, or P.T.C. For autoprothrombin
II may be combined with platelet factor 3, cal-
cium ions, and Ac-globulin to convert purified
prothrombin to biothrombin, and the latter may
be changed to citrate thrombin in 25% sodium
citrate solution. The traditional concept which
holds that prothrombin is converted only to
thrombin is not valid. At least 2 other derivatives
of prothrombin are important in biology. The dis-
covery of these derivatives and the means to
produce them not only contributes to our under-
standing of the blood clotting mechanisms, but
also describes molecular transformations quite
different from any patterns previously recog-
nized.

1152. Effect of insulin on blood levels of in-
fused pentoses in man. STANTON SEGAL,
JAMES B. WYNGAARDEN AND JOSEPH FOLEY
(introduced by Bren Buoom). Natl. Inst. of
Arthritis and Metabolic Diseases, Natl. Insts. of
Health, PHS, Bethesda, Md.

Ten to 20 gm quantities of p-xylose, p-arabinose
and L-arabinose have been infused intravenously
into normal humans over 8-30 min. periods.
When blood concentrations determined at various
time intervals following infusion were plotted on
semilogarithmic paper, straight lines were ob-
tained. The biological half-times of these sugars
ranged from 44 to 96 min., and the urinary re-
coveries from 27 to 60%, in various subjects, no
consistent differences being observed between
pentoses. When the linear semilogarithmic disap-
pearance phase was established (45-60 min.) 0.1 uv
of insulin/kg was injected intravenously. After a
latent period of 10 min. a marked decrease of
blood level of p-xylose and .-arabinose results,
which is maximal in 30 min., and represents a
change of 22-33% from the concentration ob-
tained by extrapolation of the initial curve.
After this period of change, a new linear semi-
logarithmic disappearance curve is established
whose slope is similar to that of the preinsulin
curve. Insulin causes no such change in the dis-
appearance curve of p-arabinose. The effect of
insulin on p-xylose and L-arabinose levels may be
dissociated from the hypoglycemic effect, for
glucose given intravenously following the in-
sulin did not alter the pentose response to in-
sulin. Studies are in progress with other pentoses
and hexoses in order to correlate stereochemical
configuration of the sugars with insulin respon-

FEDERATION PROCEEDINGS

Volume 16

siveness, and to elucidate the mechanism of the
reaction. The results of the studies conducted to
date in man parallel those of Goldstein et al. (Am.
J. Physiol. 173: 207, 1953) in the eviscerated,
nephrectomized dog and suggest that insulin has
affected the distribution of p-xylose and L-ara-
binose, but not of p-arabinose, in body fluids.

1153. General features of nucleotide arrange-
ment in deoxyribonucleic acids. HERMAN §,
SHaprro* aND Erwin CuarcGarr. Biochemistry
Dept., Columbia Univ., New York City.

In continuation of our previous studies of the
general aspects of nucleotide sequence in DNA
(Tamo et al. J. Biol. Chem. 203: 673, 1953; HoprEs
AND CHarGAFF. Biochim. et Biophys. Acta. In
press), a quantitative investigation of the hy-
drolytic breakdown of a variety of DNA speci-
mens was carried our under _ standardized
conditions (0.1 m H.SO,, 100°) and the rates of
liberation of the 3’,5’-diphosphates of thymidine
and deoxycytidine were determined. The ease of
detachment of these substances is markedly de-
pendent upon the nature of their neighbors in
the polynucleotide chain, as shown by kinetic
studies with model deoxy dinucleotides, such as
(I) adenylic-cytidylic, (II) cytidylic-cytidylic,
(III) thymidylic-cytidylic or cytidylic-thymidylic
(with the second member of the pairs carrying
the terminal 5’-phosphate). In each case, forma-
tion of pyrimidine nucleoside diphosphates was
initiated by breakage of the glycosidic bond of
the purine in I or, much more slowly, of cytosine
in II and either cytosine or thymine in III. The
application of these procedures to intact DNA
has given quantitative information on pyrimidine
nucleotides that are situated amidst purine
nucleotide stretches. When the degradation is
extended to 2 hr. a slower release is noted of those
diphosphates that originate from small poly-
pyrimidine units which have been formed under
these mild hydrolytic conditions. The compara-
tive study of diphosphate formation from several
unfractionated or fractionated DNA specimens
from different species shows characteristic dif-
ferences, but justifies the general conclusion that
the polynucleotide chains under comparison
consist predominantly of polypurine and poly-
pyrimidine units.

1154. Hydrolysis of a carbon-carbon bond by
trypsin. RaymMonp SHaprra* anp Davin G.
Douerty. Biology Div., Oak Ridge Natl. Lab.,
Oak Ridge, Tenn.

Studies in this laboratory have established that

a proteolytic enzyme, a-chymotrypsin, could

hydrolyze a susceptible carbon-carbon bond (J.

Am. Chem. Soc. 77: 4887, 1955). In an attempt to

extend this observation to another system,

cal

218
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March 1956

trypsin was selected since it possessed the
requisite specificity requirements, i.e. its simple
substrates are bound to the enzyme on only one
side of the susceptible bond (J. Biol. Chem. 179:
665, 1949). It was desirable to simplify the syn-
thetic approach to the 8-keto ester, therefore the
hydrolysis of methyl-6-amino-caproate by trypsin
was investigated. Contrary to a previous report
it proved to be readily susceptible to tryptic
action. Methyl-5-amino-valerate was also slowly
hydrolyzed by trypsin while methyl-4-amino-
butyrate was completely resistant. The trypsin
preparation used had no esterase activity as
evidenced by the failure to hydrolyze a series of
fatty acid esters as well as the lack of inhibition
of methyl-6-amino-caproate hydrolysis by so-
dium fluoride-treated trypsin. The desired keto
ester, ethyl-3-keto-8-amino-octanoate, was syn-
thesized from e¢ carbobenzoxy amino caproyl
chloride and ethyl acetoacetate in the usual
manner and subjected to tryptic action. The keto
ester was slowly hydrolyzed in a manner similar
to the a-chymotryptic hydrolysis of ethyl-5-(p-
hydroxyphenyl)-3-keto valerate to yield « amino
caproic acid.

1155. Metabolism of DOPA. Kennetu N. F.
SHaw,* ARMAND McMiILuaAn* anp Marvin D.
ArmstroneG. Lab. for Study of Hereditary and
Metabolic Disorders, Univ. of Utah, Salt Lake
City.

Urine from all normal humans contains a small
amount of a substance which is chromatographi-
cally identical with homovanillic acid (HVA)
(ARMsTRONG, SHAW AND WALL. J. Biol. Chem.
218: 293, 1956) and which appears to be of endog-
enous origin, since it is excreted in the same
amount (3-8 mg/day) by humans eating a syn-
thetic diet. Booth et al. (Federation Proc. 14:
321, 332, 1955) reported that rabbits excreted
HVA and increased amounts of m-hydroxyphenyl-
acetic acid (m-HPAA) after the ingestion of
homoprotocatechuic acid (HPA); all 3 acids were
excreted after the ingestion of DOPA. The me-
tabolism of DOPA, 3-methoxy-pL-tyrosine, HPA
and HVA in the human has been studied, by the
use of paper chromatography, to examine urine
for metabolites. After the ingestion of 500 mg of
t-DOPA, HVA (35%) and a substance having
the chromatographic properties of HPA (45%)
appeared in the urine. After the ingestion of 250
mg of HPA, a similar excretion pattern of HPA
and HVA was found, and after 250 mg of HVA,
75% appeared in the urine unchanged. No ap-
preciable amount of m-HPAA could be detected
in the urine after the administration of any of
these compounds. When pt-DOPA was ingested,
a substantial amount of the amino acid (pre-
sumably the p-form) was also excreted. In con-

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

353

trast to the high recoveries cited above, only
4% of ingested 3-methoxy-tyrosine could be ac-
counted for in the urine in the form of HVA.

1156. Coexistence of insulin-responsive and
insulin-nonresponsive glycolytic systems
in rat diaphragm. W. N. SHaw* anp W. C.
Stapie. John Herr Musser Dept. of Research
Medicine, Univ. of Pennsylvania, Philadelphia.
Repeated observations have shown that the

aerobic formation by rat diaphragms in vitro of
lactate from U-C"-glucose in a 0.05 m phosphate-
saline medium, px 7.4, is unaffected by the addi-
tion of insulin. In contrast, glycogen synthesis is
always increased by insulin. A chromatographic
separation of the Embden-Meyerhof intermedi-
ates from the diaphragm showed that insulin
increased the conversion of glucose to glucose-6-
phosphate, glucose-l-phosphate and glycogen,
but had no effect on fructose-1,6-diphosphate or
pyruvate as well as lactate. These conclusions
are based on the total stoichiometric quantita-
tion and the determination of the specific activity
of the intermediate compounds. Further experi-
ments indicate that there is a free interchange
between the medium and the intermediates in
the case of the nonresponsive pathway (lactate
synthesis) but not in the case of the responsive
pathway (glycogen synthesis). We have concluded
that under our. experimental conditions 2 gly-
colytic pathways are operative in the diaphragm:
1) a glycogen-synthesizing pathway which is
responsive to insulin, 2) a lactate-synthesizing
pathway which is nonresponsive to insulin. Pre-
sumably these systems are identical except for
this difference in insulin responsiveness. These
data appear to be in harmony with the concept
that a transport mechanism is involved in the
insulin-responsive system but is not in the non-
responsive system.

1157. Application of PDR amino acid index to
measurement of net utilization of heated
proteins. A. LEONARD SHEFFNER, RICHARD
Apacui* AND Harry Spector. Quartermaster
Food and Container Inst. for the Armed Forces,
Chicago, Ill.

The pepsin digest-residue (PDR) amino acid
index (Federation Proc. 14: 279, 1955) was deter-
mined for several proteins subjected to a variety
of heat treatments. The results indicated that the
PDR index accurately reflected the effects of heat
processing upon net protein utilization (biological
value X digestibility). Division of the PDR index
by the respective coefficients of digestibility
yielded values which corresponded to the biologi-
cal value as determined by nitrogen balance in
rats. In the case of raw soybean meal the PDR
index overestimated the rat assay value. In this

354

respect, data are presented which indicate that
when raw soybean meal is previously digested with
pepsin the antitryptic factor suppresses the subse-
quent in vitro release by pancreatin of cystine and
lysine to a much greater extent than any of the
other essential amino acids. However, biological
values (rat assay) obtained with raw and heated
soybean meals supplemented to overcome de-
ficiencies in these amino acids showed that the
beneficial effect of heating was approximately the
same for both the supplemented and unsupple-
mented soybean meals. Consequently, the dis-
crepancy between the PDR index and the net
protein utilization value of raw soybean meal is
interpreted as being due to the presence in the raw
soybeans of toxic substances which exert effects
apparently unrelated to the digestion of protein.

1158. Thromboplastic cell component of
human blood. GeorGE Y. SHinowaRa. Dept. of
Pathology, College of Medicine, Ohio State Univ.,
Columbus.

It was previously demonstrated that human
blood cells contain a lipoprotein which together
with a globulin in plasma fraction I (Cohn) will
completely convert prothrombin into thrombin in
the presence of ionic calcium alone. This lipo-
protein, then identified as thromboplastic cell
component (TCC), made possible a coagulation
mechanism based on blood factors only, i.e. with-
out tissue thromboplastin (J. Lab. & Clin. Med.
38: 11, 1951). By modification of the original
differential ultracentrifugation procedure, the
purity of the lipoprotein preparation was increased
from 0.18 to 29.28 TCC units/ug solid. Electro-
phoretic analyses of the highly purified prepara-
tions demonstrated a mobility in excess of human
albumin and one boundary. However, electron
microscopy indicated heterogeneity. Ultraviolet
studies revealed a maximum absorption at 250 yu.
Chemical analysis disclosed the following in gm/
100 gm solid: total nitrogen, 6.58; lipid nitrogen,
1.35; lipid phosphorus, 2.19; free cholesterol,
30.78, and ester cholesterol, 0.00. In another series
of experiments, TCC activities of various blood
cell fractiong were measured: platelets (99.3-
99.4%) were found to contain 0.73 u/million cells;
erythrocytes (99.3-99.8%), 3.88 u/million cells.
Normal leucocytes could not be associated with
TCC activity. Although more than 98% of the
TCC lipoprotein is associated with erythrocytes,
the remainder, though slight, was shown to have a
significant role by experiments in which this and
other coagulation factors were quantitatively
determined during the spontaneous clotting of
normal blood.

1159. Formation of radioactive polyenes and
carotenoids in ripening tomatoes. ELIE A.

FEDERATION PROCEEDINGS

Volume 16

SHNEOUR* AND IRviING ZABIN. Dept. of Physio-
logical Chemistry, Univ. of California Med. Ctr.,
Los Angeles.

Tomatoes which were removed from the plant
and which had just begun to turn red were injected
with’ methyl-labeled acetate. After a ripening
period of 5-12 days, a number of polyenes and
carotenoids were isolated, purified and assayed
for radioactivity. The following results were ob-
tained: lycopene had a specific activity of 2,500
c/min/mg C, beta-carotene 800, phytofluene 700,
and a fraction consisting of phytoene and tetra-
hydrophytoene 450 c/min/mg C. The Porter-
Lincoln hypothesis for carotene biosynthesis,
which suggests successive dehydrogenations of
C-40 colorless polyenes leading to the formation of
colored carotenoids, has received wide support.
This sequence involves the intermediary formation
of tetrahydrophytoene, phytoene, phytofluene,
zeta-carotene, tetrahydrolycopene and lycopene.
While our results cannot as yet rule out the Porter-
Lincoln mechanism, the data obtained here do
not offer any support for this scheme. (Supported
by a research grant from the Natl. Insts. of
Health, PHS.)

1160. Glucogenesis from 1-C'!-acetate in non-
diabetic and diabetic man. WALTON W.
SHREEVE. Med. Dept., Brookhaven Natl. Lab.,
Upton, N. Y.

Subsequent to a carbohydrate fast for 15-42 hr.,
100-200 ue of 4-C'*-acetate with 1.0 mm acetate
carrier were administered intravenously to each of
3 cancer patients (considered to have normal
carbohydrate metabolism) and 2 diabetic patients.
The diabetics (11- and 38-yr.-old white females)
were of the ‘juvenile’ type, insulin-free and in
marked ketosis and acidosis. From blood collected
2 hr. after C' injection and from diabetic urine
were isolated specimens of glucose, which was de-
graded by several means so as to permit measure-
ment of radioactivity in various individual or

groups of carbon atoms. In accord with various’

animal studies, the C'* in blood glucose from both
nondiabetic and diabetic human beings was lo-
cated almost entirely in the 3 and 4 carbon po-
sitions. In nondiabetic subjects the activity in
these two carbons was approximately equal; in
diabetic subjects there was 10-50% more activity
in carbon 4 than in carbon 3. The 1, 2, 5 and 6
carbons in all cases contained only trace amounts
of C'* (0.5-3% of the total glucose count in each),
and no definite pattern was detectable. The find-
ings are in accord with the concept of glucose
formation essentially by the action of the Krebs
cycle, and/or CO: fixation, and reversal of Emb-
den-Meyerhof glycolysis. There was no indication
of the utilization in diabetes of other pathways
which might result in significant net formation of

ei go

ume 16

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March 1956

glucose from fat. (Supported by the Atomic
Energy Commission.)

1161. Pyruvic dehydrogenase system of Clos-
tridium pasteurianum. A. L. SHuG anp P. W.
Witson. Dept. of Bacteriology, Univ. of Wiscon-
sin, Madison.

Cell-free preparations of this organism in the
presence of inorganic phosphate rapidly ferment
pyruvate to acetyl phosphate, carbon dioxide and
hydrogen. Enzymes catalyzing this reaction did
not sediment on 30-min. exposure to 144,000 X g.
Manometric and spectrophotometric tests showed
that methyl viologen, benzyl viologen, methylene
blue and cytochrome c serving as electron ac-
ceptors retarded hydrogen evolution from py-
ruvate. DPN and TPN are not reduced nor do they
stimulate H. evolution from pyruvate. These
preparations had a marked dilution effect with
respect to hydrogen evolution; however, complete
restoration of activity was obtained by the ad-
dition of Fe+*+, MoO;, and methyl] viologen. Spec-
trophotometric tests indicated that methyl
viologen and endogenous flavin were reduced
prior to Hz: evolution and that Fe** stimu-
lated the reduction of methyl viologen, whereas
MoO; increased hydrogen production from the
reduced dye. Antimycin, 5 X 107? uM, (preincu-
bated with 3 mg of protein) completely inhibited
the reduction of methyl viologen and H: evolution,
but did not inhibit CO: production. The same
concentration of antimycin had no effect on the
oxidation of H2 by methyl viologen or the evolu-
tion of hydrogen from the reduced dye. Carbon
monoxide (50% v/v of gas phase) completely in-
hibited reduction of methyl viologen by pyruvate
or hydrogen; however, the inhibition of hydrogen-
ase could be reversed by the removal of CO by
evacuation. Nitric oxide (1 and 3% v/v of gas
phase) completely and irreversibly inhibited hy-
drogen evolution and reduction of methy] viologen
by pyruvate of Hz. KCN (10-* m) completely in-
hibited the reduction of methyl viologen by
pyruvate, but only partially inhibited hydrogen-
ase activity. The ability to evolve hydrogen from
pyruvate was abolished by heating at 60°C for 10
min. Conversely, fractionation with (NH,4)2SO,
gave preparations which produced hydrogen from
pyruvate only when supplemented with purified
hydrogenase.

1162. Glucose-1-phosphate transphosphoryl-
ase. J. B. Stppury, Jr.,* Lawson RoOSENBERG*
AND Victor A. Nassar. Dept. Pediatrics, Johns
Hopkins Hosp., Baltimore, Md.

A glucose-l-phosphate transphosphorylase has
been demonstrated in rabbit muscle which cata-
lyzes the reaction whereby 2 m of glucose-1-phos-
phate form 1 m each of glucose-1,6-diphosphate

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

355

and glucose. It was found chiefly in the ammonium
sulfate fraction between 0.5 and 0.7 saturation.
The forward reaction is stimulated by Mg**, has
a pH optimum of 7.6 and it is unaffected by ATP
in concentrations up to 0.25 uM/ml. It was un-
affected by cysteine, adenylic acid, Cut*+ 10°‘ m
NaF 4 X 107°, and Mn did not stimulate diphos-
phate formation. The reverse reaction was in-
hibited by added Mg**. The findings in this re-
action suggest a novel mechanism for the role of
Mg* in that it favors the forward reaction by
complexing with the glucose-1,6-diphosphate and
thus in effect removing it from the reaction.

1163. Isocitric dehydrogenases from heart
muscle. GUNTHER SIEBERT AND JEAN Dusuc
(introduced by G. W. E. Puaut). Dept. of Bio-
chemistry, New York Univ. College of Medicine,
New York City.

Sodium versenate extracts of acetone powders
(0.001 m) of beef or pig heart washed residue
(PLauT AND Suna. J. Biol. Chem. 207: 305, 1954)
exhibit a 4-12-fold higher specific activity of TPN -
linked isocitric dehydrogenase (500-1800 u/mg)
than previous preparations made from whole
tissue acetone powders. It has been possible to
achieve a 4-6-fold purification of the TPN-linked
enzyme from such extracts by means of ammonium
sulfate fractionation and calcium phosphate ad-
sorption-elution or by cation exchange procedures,
yielding specific activities up to 4000 u/mg pro-
tein. Although this is not a high level of purifica-
tion over the activity of the original extract it
is 2.7 times higher than that of Moyle and Dixon
(Biochim. et Biophys. Acta 16: 434, 1955) who re-
ported a specific activity of 1500 u/mg protein for
a 90% pure (electrophoresis) preparation from pig
heart. The TPN-linked isocitric dehydrogenase at
the present state of purification can still carry out
the reoxidation of reduced TPN in the presence of
a-ketoglutarate and CO:. DPN-linked isocitric
dehydrogenase can be purified from versene ex-
tracts of pig heart washed residue preparations by
the same procedure as that used for beef heart
(PLaut AND Sune, Meth. Enzymol. 1: 710), achiev-
ing about the same level of purification and yield.
This preparation is essentially free of TPN iso-
citric dehydrogenase activity.

1164. Enzymes associated witha mitochondrial
membrane fraction. PHILIP SIEKEVITZ AND
MicuaEt L. Watson.* Rockefeller Inst., New
York City.

Rat liver mitochondria isolated in 0.44 m sucrose
when disrupted with 0.3% Na deoxycholate lose
their matrix, and 80% of their proteins become
solubilized. Mitochondrial suspensions so treated
were centrifuged differentially and the pellets ob-
tained analyzed biochemically and examined in

356 FEDERATION PROCEEDINGS

the electron microscope. The final pellet (60 min
at 100,000 X g) consisted of membranous com-
pound vesicles. The derivation of these elements
from mitochondrial membranes could be ‘traced
from cross-sections of mitochondrial pellets
layered under Na deoxycholate which showed all
stages of disruption from intact mitochondria to
compound vesicles. Derivation from contaminat-
ing microsomal membranes could thus be ruled
out. The final pellet contained 5% of the pro-
tein-N, 15% of the phospholipid-P, 25-35% of the
succinoxidase and 35-55% of the cytochrome
oxidase activities of the original mitochondria.
DPNH-cytochrome c¢ reductase and adenylate
kinase were solubilized by this treatment, while
coupled phosphorylation was completely in-
hibited. Localization of enzymes of the Krebs
cycle other than succinoxidase could not be defi-
nitely determined because of marked inhibition by
deoxycholate. The succinoxidase of the mito-
chondria and of the final pellet responded simi-
larly to activation by Al*** and Ca** and to in-
hibition by antimycin A. After washing, 90% of the
recoverable cytochrome oxidase remained in the
pellet. The final pellet contained in addition some
cytochrome c, and since it exhibited a 3-fold con-
centration of phospholipid and approximately
5- and 7-fold concentrations of succinoxidase and
cytochrome oxidase over the intact mitochondria,
it is possible that the succinoxidase complex
resides on, or is part of, the mitochondrial mem-
brane.

1165. ‘Labile citrovorum factor’ in human
urine. M. SILVERMAN, F. G. EBauGu* ann R. C.
GaARDINER.* Natl. Insts. of Health, PHS, Be-
thesda, Md.

The bulk of the citrovorum factor activity ex-
creted in human urine by subjects who have in-
gested folic acid is lost after several hours of
storage in air at pH 7.0 and 23° (NicHoL et al.
Science 121: 275, 1955; DropHar et al. Federation
Proc. 14: 201, 1955). By autoclaving in the presence
of ascorbate, the factor responsible for this ac-
tivity can be converted into CF. Active fractions
have been obtained from urine by 1) precipitation
of the material at px 2.5 and 0°, 2) extraction of the
precipitate with dilute HCl at 0°, 3) adsorption of
the activity from the acid solution on charcval at
0°, 4) elution under He with ascorbate, ethanol,
ammonia solutions, 5) partition chromatography
of the concentrated eluates on Sulka-flok (butanol-
formic acid at 23°) and 6) gradient elution from
Dowex-1 formate at 0°. All concentration pro-
cedures were carried out under reduced pressure
in an atmosphere of He. Fractions from Dowex-1
have a ratio of Streptococcus faecalis to Leuconostoc
citrovorum growth activity of 1. Lyophylized
fractions obtained from Dowex-1l, possess 50% or

Volume 15

more CF activity. These products markedly re-
semble anhydrocitrovorum factor (N*-N” bridge
compound) in that they are readily converted to
CF on heating under He or in ascorbate solution.
Also, under anaerobic conditions in 0.1 N HCl,
the products possess an absorption peak at 355-
360 u. On oxidation, both the 355-360 » peak and
growth activity for Leuconostoc citrovorum are
lost.

1166. Enzymatic degradation of deoxyribo-
nucleic acid containing 5-bromouracil.
Rosert L. StnsHermer. Dept. of Physics, Iowa
State College, Ames.

A deoxyribonucleic acid (DNA) in which a
portion of the thymine residues have been re-
placed by 5-bromouracil has been prepared from
a thymine-requiring strain of Z. coli grown in the
presence of 5-bromouracil, as described by S.
Zamenhof (Nature 174: 307, 1954) This DNA has
been enzymatically degraded and the products
fractionated by ion exchange chromatography.
The separation and distribution of the products
have been studied.

1167. Factors modifying amino acid inter-
relationships and related peptide effects in
lactic acid bacteria. Ropert J. SrRNY AND
Joun N. Mitts (introduced by V. G. HELLER).
Dept. of Agricultural Chemistry, Oklahoma Agri-
cultural Exp. Station, Stillwater.
Interrelationships between amino acids and bac-

teria are well known, effects of pH on certain of

these interrelationships have been reported by
various workers and the inhibition of valine utili-
zation by Lactobacillus arabinosus has been shown
to be greater with high Na* in the medium. Further
investigations of these effects reveal that in L.
arabinosus several different factors modify the
degree of inhibition of valine utilization by either
high leucine or isoleucine. These factors include
initial pH, the presence or absence of NH,*, and
the relative amounts of Nat and K* in the medium.

No general statements can be made here regarding

these interrelated effects, but the variations in the

inhibition observed under different specific con-
ditions will be presented. In a different type of

interrelationship in Leuconostoc mesenteroides P-60

(J. Biol. Chem. 190: 547, 1951), the degree to which

proline utilization is improved by high arginine

in the medium has also been found to be markedly
affected by the same factors. Similarly, the favor-
able effect of high proline or arginine utilization
and of high arginine on glycine utilization is also
altered in degree by these factors. Since in this
latter interdependence, peptide-bound glycine is
more readily utilized than is free glycine (Feder-
ation Proc. 13: 298, 1954), the effects of these
factors on the utilization of peptide-bound glycine

= 2S = 2. @& Gee oo tet ot & 2 = A aot ot & fe wee OF a aCe elt

_- oo fo —-

f--]

—

ne 15

y re-
ridge
ed to
ition.

HCl,

c and
» are

ribo-
acil.
Towa

ch a
n re-
from
n the
yy S.
A has
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aphy.
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nter-
ts in
" AND
LER).
Agri-

1 bac-
iin of
d by
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rther
in L.
y the
»ither
clude
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; P-60
which
rinine
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lycine

March 1966

will also be considered. (Supported in part by
Research'Grant G-514, Natl. Science Fndn.)

1168. Human metabolism of 1-dehydro-17-
hydroxycorticosteroids (A’-17-OHCS). W.
Roy SLAUNWHITE, JR.* AND Avery A. SAND-
BERG* (introduced by C. CarruTHers). Ros-
well Park Memorial Inst., Buffalo, N. Y.

The metabolism of 1-dehydrocortisone and
1-dehydrocortisol was compared with that of
cortisone and cortisol in patients of both sexes
between the ages of 12 and 75 yr. Both oral in-
gestion (40-240 mg/day) and intravenous injection
(1 mg/kg b. wt.) were employed. The rate of dis-
appearance of intravenously injected A’-17-OHCS
from the plasma was indistinguishable from that of
17-OHCS. Ultrafiltration experiments with steroid
added in vitro to normal plasma demonstrated
that A’-17-OHCS are loosely bound to plasma pro-
teins to approximately the same degree as 17-
OHCS. Urine analysis revealed that not only was
there no conversion of A’-17-OHCS to 17-ke-
tosteroids (17-KS), but administration in large
amounts inhibited endogenous production of
17-KS. The urinary excretion of unconjugated
Porter-Silber chromogens was greatly enhanced in
the case of A’-17-OHCS at the expense of the con-
jugated chromogens. When 4-C'*-cortisol was
administered simultaneously with 240 mg 1-dehy-
drocortisol, each compound was metabolized inde-
pendently of the other. That is, in spite of the
overwhelming amount of 1-dehydrocortisol, the
radioactive tracer was converted to 17-KS and con-
jugated with glucuronic acid in the same amounts
as when it was administered with 240 mg of carrier
cortisol. Work is continuing on the isolation and
identification of the urinary excretory products
and on the mechanism of action of A’-17-OHCS.

1169. Transmethylation studies involving
homocysteine as methyl acceptor. N. H.
StoaNnE, E. M. Bogerano,* B. CouLoms* AND
B. L. Hutcuines. American Cyanamid Co., Re-
search Div., Lederle Labs., Pearl River, N.Y.
Betaine-homocysteine transmethylase of pigeon

liver extract: Attempts to split the enzymatic ac-

tivity of the protein into coenzyme and apoenzyme
were unsuccessful. There was no resolution of the
enzyme in the extract or in the purified state by
exhaustive dialysis over wide ranges of pH, dialysis
against versene, treatment with ion exchange
resins or adsorbents or acid precipitation. The
enzyme can be purified 20-fold by precipitation of
the cell-free extract with (NH,)2SO, (50% satura-
tion) and heat treatment of the precipitated pro-
tein (50 mg/ml) at 74° for 120 sec. The apparent
energy of activation of the enzyme-substrate com-

plex was determined to be 19,000 cal. between 23

and 37°. Attempts to dissociate the rat liver en-

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS 357

zyme by the method of Ericson et al. (J. Biol.
Chem. 212: 537, 1955) were unsuccessful. Dimethyl-
thetin (DMT)-homocysteine transmethylase: The
pigeon liver extract contains an active DMT-
transmethylase (SLOANE et al. Federation Proc. 14:
282, 1955). DMT is approximately 15 times more
efficient as a methyl donor than betaine at equiva-
lent concentrations of substrate and enzyme at pH
7.5. The DMT enzyme is also stable to heat treat-
ment. The end products of the reaction are not
inhibitory since stoichiometric amounts of methi-
onine are formed from DMT; the data show a
transfer of 1 methyl/m of DMT. Dimethylglycine,
which has been shown to competitively inhibit
betaine transmethylase, similarly inhibits the
enzymatic activity with DMT as methyl donor.
Inhibition by thioglycollate of both transfer re-
actions is overcome by addition of excess of either
substrate. The pigeon liver extract is unable to
transfer the thiomethyl group of thiomethyl-
adenosine to a-aminobutyric acid, nor can it cata-
lyze the cleavage of pu-cystathionine to homo-
cysteine at pH 7.5.

1170. Respiration and phosphorylation in ex-
tracts of Rhodospirillum rubrum. LucILE
SMITH AND MARGARETTA BALTSCHEFFSEY (intro-
duced by D. R. Gopparp). Johnson Research
Fndn., Univ. of Pennsylvania, Philadelphia.
Respiration and photosynthetic phosphoryla-

tion were studied in extracts of Rhodospirillum

rubrum, along with accompanying spectrophoto-
metric changes. The extract was prepared by
grinding with alumina. It showed oxygen uptake on
addition of several substrates; with succinate the
Qo,/protein was about 15 at 26°C. Illumination in
the presence of ADP resulted in the disappearance
of as much as 40 um of inorganic phosphate/hr.
times O.D. 800 my, with or without added sub-
strates. Disappearance of inorganic phosphate in
the dark, with or without substrates and oxygen,
was very small or zero. Photosynthetic phos-
phorylation was completely inhibited by 10 m
3-hydroxy,1-heptylquinoline, while oxygen up-
take was unaffected. The difference between the
spectrum of the extract in the anaerobic state and
that in the aerobic state was found to be similar to
that of the whole cells. However, illumination of
the extract in either the aerobic or the anaerobic
state resulted in the appearance of a peak in the
absorption spectrum at about 434 my; a similar
absorption peak resulted from addition of sodium
hydrosulfite to the anaerobic extract. The ab-
sorption peak did not appear when the anaerobic
extract was illuminated in the presence of ADP,
except in the presence of the inhibitor of phos-
phorylation. The results suggest that the system
in Rhodospirillum rubrum extract which is re-
sponsible for photosynthetic phosphorylation may

358

be different from that of the respiratory chain.
The substance having an absorption band at 434
my appears to dominate the system involved in
photosynthetic phosphorylation.

1171. Mechanism of calcification. ALBERT
Epwarp Soper, Martin BurcGer,* JosEePH
SaMACHSON* AND NorMAN Stovik.* Dept. of
Biochemistry, Jewish Hosp. of Brooklyn, N.Y.
Bones were demineralized with ethylenediamine

tetra-acetic acid (EDTA). When treated with

chondroitin sulfate, followed by calcium chloride,
such bones remineralized in a calcifying solution.

A second method of inducing mineralization was

to treat bones with calcium chloride, followed by

sodium phosphate. When the order of treatment
was reversed, mineralization did not take place.

From these studies and from our studies with in-

hibitors, the following picture of the calcifying

mechanism emerges. The calcium-collagen-chon-
droitin sulfate complex is able to initiate the
nuclei of crystallization. These nuclei undergo
crystal growth to form apatite. Treatment with
calcium followed by phosphate produces these
nuclei and thus remineralization can take place
without the nuclei-producing mechanism which
appears to involve a collagen chondroitin sulfate
complex. The collagen appears to be a specific
protein. (Supported by the Natl. Inst. for Dental

Research, Natl. Insts. of Health, PHS, and a

contract between the Office of Naval Research,

Dept. of the Navy and the Jewish Hosp. of

Brooklyn, NR 180 025.) (Reproduction in whole or

in part is permitted for any purpose of the United

States Government.)

1172. Partial purification and _ preliminary
analyses of certain soluble azoproteins from
livers of rats fed 3’-methyl-4-dimethyl-
aminoazobenzene. Sam Soror, Marityn G.
Orr* aNp Emrty M. Youna.* Lankenau Hosp.
Research Inst. and Inst. for Cancer Research,
Philadelphia, Pa.

Previous studies appear to indicate the involve-
ment of the soluble liver ‘h’ azoproteins in amino-
azo dye hepatocarcinogenesis in the rat through
the hypothetical ‘protein deletion’ mechanism of
Miller and Miller (Adv. in Cancer Research 1: 339,
1953). Adult Lankenau-Wistar rats of both sexes
were fed ad libitum for 18-21 days diet no. 3
(MILLER AND MiLiER) including 0.057% 3’methyl-
4-dimethylaminoazobenzene and 1.0 mg _ ribo-
flavin/kg. Control rats were fed the same diet
without dye. Using free boundary electrophoresis
with a convection barrier (J. Am. Chem. Soc. 76:
4740, 1954), 100-300-mg quantities of the following
2 fractions have been isolated in each separation
from 6 resolved ‘h’ subcomponents: 1) 100% ‘h;’,
2) 3 ‘h’ subclasses: 57% ‘hs’; 30% ‘slow hz’; 138%

FEDERATION PROCEEDINGS

Volume 16

‘middle he.’ These represent 7.3%, 3.4% and 3.5%
respectively, of the soluble liver proteins of the
dye-fed rats. The ‘h;’ proteins contain 14% of all
the soluble bound dyes. Assuming the absence of
bound dyes with the minor constituent (‘middle
he’) in fraction 2, 42% of all the soluble bound
dyes are with the same subclass (‘slow he’) which
greatly increases during azo dye preneoplasia
(Proc. Am. Assoc. Cancer Research, 1956. In press).
The ‘h’ proteins of fraction 2 from control or dye-
fed rats do not contain significant amounts of non-
dialyzable nucleic acids or riboflavin. This is of

interest considering the roles of nucleic acids in’

growth and the inhibitory effect of dietary ribo-
flavin on certain azo carcinogens.

1173. Automatic recording apparatus for use
in chromatography of amino acids. DARREL
H. SpackMANn,* Wiii1amM H. STEIN AND STAN-
FORD Moore. Rockefeller Inst. for Med. Research,
New York City.

A recording apparatus has been designed to be
employed in conjunction with columns of ion ex-
change resins. The influent buffer, freed of air, is
pumped through the column at a constant rate of
30 ml (or less)/hr. by a stainless steel pump. The
effluent from the column, flowing through 1 mm
capillary tubing, is met by a stream of ninhydrin
reagent delivered at half the rate of the effluent
by a second pump. The color is developing by
passing the mixture of reagent and effluent through
a 50-ft. spiral of 1-mm bore Kel-F tubing immersed
in a boiling water bath. The optical density of the
resulting solution is measured as it flows through
a cylindrical glass cell. Three photometer units
are used, each consisting of a light source, lens,
interference filter and photovoltaic cell. Two of
the filters transmit maximally at 570 mu, and the
third transmits at 440 my, to permit the determina-
tion of proline and hydroxyproline. The effective
diameter of the glass cell is reduced from 2.2 to 0.8
mm as it passes through one of the units contain-
ing a 570 my filter in order to extend the range of
the instrument when the optical density exceeds
1.3 with the larger cell. The optical density meas-
ured by each photometer is recorded by a Leeds
and Northrup three-point recorder. The published
ion exchange procedure using an 8% cross-linked
resin (Moork AND STEIN. J. Biol. Chem. 192: 663,
1951) has been simplified to permit a complete
amino acid analysis of a peptide or protein hy-
drolysate in 20 hr., using a single operating tem-
perature (50°) and 3 eluents for the 2 columns. The
accuracy is 100+3%.

1174. Relationship of nitrite and hydroxyl-
amine reductases to nitrate assimilation
and nitrogen fixation in Azotobacter.
DonaLp SpENCER,* Hasime TAKAHASHI* AND

kt: es”

ia ote Ge ote lOO Cr

pow

ume 16

d 3.5%
of the
® of all
ence of
middle
bound
| which
oplasia
press).
or dye-
of non-
is is of
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y ribo-

or use
JARREL

STAN-
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1 to be
ion ex-
air, is
rate of
p. The

1 mm
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ing by
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- of the
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r units
», lens,
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nd the
rmina-
fective
2 to 0.8
ntain-
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xceeds
- meas-

Leeds
ylished
-linked
2: 663,
mplete
in hy-
g tem-
is. The

roxyl-
lation
acter.
(* AND

March 1956

Atvin Nason. McCollum-Pratt Inst., Johns

Hopkins Univ., Baltimore, Md.

A soluble extract obtained by sonic oscillation
of nitrate-grown Azotobacter vinelandii catalyzes
the reduction of nitrite and hydroxylamine to
ammonia by reduced pyridine nucleotides. The
rate of reduction of nitrite is 3 times as fast as
that of hydroxylamine. The nitrite and hydroxyl-
amine reductases, which have been purified ap-
proximately 5-fold, are different enzymes, as indi-
cated by the adaptation experiments below. More-
over, nitrite reductase activity is stimulated
more than 2-fold by flavin-adenine-dinucleotide
and only slightly by the mononucleotide, whereas
hydroxylamine reductase is inhibited by these
flavins. Inhibition by metal-binding agents indi-
cates a metal component in both enzymes. Ap-
proximately 1 m of ammonia is formed for each
mole of nitrite reduced. Nitrite reductase is an
adaptive enzyme system whose formation is in-
duced by nitrate but not by Ne or glutamate. Al-
though hydroxylamine reductase activity is the
same in extracts of cells grown on No, ammonium
sulfate or glutamate, there is 3-4 times as much
enzyme in extracts of cells grown on nitrate. Com-
parable growth is obtained on each of the nitrogen
sources. The above adaptation results support the
role of nitrite and hydroxylamine as possible
intermediates in nitrate assimilation. The data
also suggest, however, that the pathway of N--fix-
ation does not involve the oxidation of N: to nitrite
or nitrate before ultimate reduction to the amino
form as suggested by others; and that hydroxyla-
mine is probably not an intermediate in N: fixation
in view of the failure of increased formation of
hydroxylamine reductase in N2-grown cells.

1175. Unsaponifiable fraction of brain lipides.
WarRREN M. Sperry. Depts. of Biochemistry,
New York State Psychiatric Inst. and College of
Physicians and Surgeons, Columbia Univ., New
York City.

The digitonin-precipitable sterols of brain un-
saponifiable fractions, obtained as previously de-
scribed (Federation Proc. 14: 284, 1955), were de-
termined by a gravimetric procedure which gave
quantitative recovery of pure cholesterol. The
amounts of sterols, either calculated as cholesterol
from the digitonide weights, or determined directly
by isolation from the digitonides by the pyridine-
ether method (ScHOENHEIMER, R. aNnp H. Dam.
Zischr. f. physiol. Chem. 215: 59, 1933), were about
5% higher than the amounts of cholesterol deter-
mined by the Sperry-Webb method (J. Biol. Chem.
187: 97, 1950). The difference is presumed to repre-
sent saturated sterols. About } of the unsaponifi-
able lipides was not precipitated by digitonin.
About 3 of this weight was extracted by ether from
the dry residue of the mother liquor from the digi-

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

359

tonin precipitation. The remainder was recovered
by application of the pyridine-ether method to the
extracted residue. The latter fraction is presumed
to represent sterols whose digitonides are soluble
under conditions which give quantitative precipi-
tation of cholesterol. From nitrogen determina-
tions, these fractions cannot contain more than 3%
of sphingosine. (Supported in part by a grant from
the Natl. Multiple Sclerosis Society.)

1176. Enzymatic action of fluids from cystic
brain tumors. II. M. Sprecet-Apour anp H.
T. Wycis.* Temple Univ. School of Medicine and
Hosp., Philadelphia, Pa.

Continuing studies have been presented before
this Society (Federation Proc. 13: 301, 1954; J.
Neuropath. 12: 601, 1954). The results obtained in
9 new cyst fluids and 6 controls are reported here.
The material comprises 3 cases of craniopharyn-
gioma, the clinical aspects of which have been
described (Confinia neurol. 14: 193, 1954), 2 meta-
static carcinomas and 4 gliomas of various histo-
logical properties. As controls cyst fluids were
used from 3 benign central nervous system tumors
and cerebrospinal fluid from 3 malignant growths
in the brain. The technique of detecting enzymatic
properties by ultraspectrographic methods and
the substrates, nucleic acids and their basic de-
rivatives, were the same as used in former publi-
cations. Among the craniopharyngeal tumors 1
which has been treated with radiophosphorus for
nearly 2 yr. and was clinically improved showed a
marked drop in the enzymatic power of the cystic
fluid. The metastatic tumors gave various findings
according to their histology. In the group of
gliomas there was as noted previously a certain
parallelism between the height of the enzymatic
power of the cyst fluid and the histologically indi-
cated malignancy. Among the controls only the
cerebrospinal fluid of the ependymoma had marked
enzymatic power. This may be explained by the
site of the tumor. The enzymatic power of the
cystic fluids decreases at a lower rate at —5° but
the decline is different for various substrates. A
collodion ultrafilter that holds back hemoglobin
is permeable for enzymatic active substances.
Fractionation of fresh cyst fluid with (NH,4)2SO,
seems to indicate that the enzymatic action is
mostly in the 3 saturation fraction.

1177. Inhibition of pyruvate metabolism in-
duced by virus infection. JoHN Sp1z1zEN
(introduced by L. PrttemMER). Dept. of Micro-
biology, Western Reserve Univ., School of Medi-
cine, Cleveland, Ohio.

Little is known about metabolic alterations oc-
curring as a result of virus infection. We have ob-
served that infection of cells of Escherichia coli
with virus T; causes an immediate and complete

360

inhibition of pyruvate utilization both aerobically
and anaerobically. Inhibitors of viral adsorption,
such as specific antiserum, prevent this block in
pyruvate metabolism, indicating a direct action of
the virus. When host cells are grown and infected
in synthetic media containing lactate or /-serine
(but not d-serine) as carbon source, viral infection
results in a rapid and progressive accumulation of
pyruvate under aerobic conditions (also anaero-
bically with l-serine). Viral replication can occur
optimally under conditions where pyruvate me-
tabolism is completely blocked. On the other
hand, with virus T2, there is an initial block in
pyruvate metabolism; subsequently, under condi-
tions optimal for viral proliferation, the cells
partially recover their ability to oxidize pyruvate.
Adsorbable, but not infectious, ultraviolet ir-
radiated virus as well as the protein moiety of T:
(‘ghost’) also inhibits pyruvate metabolism. This
indicates that the effect of virus on pyruvate
utilization is associated with the initial processes
of viral attachment. Since pyruvate accumulates
when the substrate is lactate or l-serine, altered
permeability appears to be excluded as an explana-
tion of the block in pyruvate metabolism.

1178. Tolerance to arsanilic: acid in mink.
Henry C. Sprutu,* Rosert A. RigeKER* AND
Dovetas V. Frost. Abbott Labs., North Chicago,
Til.

Ten female and 3 male, adult, healthy mink were
fed a standard ranch diet containing 0.005%-0.02%
arsanilic acid in the dry diet. The study was con-
ducted over a 6-month period and included the
breeding, whelping and weaning periods. These 13
mink, including 4 mutations, were divided into 3
family groups. Group 1 was started on 0.005%,
group 2 on 0.01% and group 3 on 0.02% arsanilic
acid of the dietary solids. The animals were bred.
Normal litters were born 3-10 wk. after arsanilic
acid feeding was begun. A separate concurrently
run experiment indicated that 0.05%-0.1% dietary
levels of arsanilic acid were tolerated. The ar-
sanilic acid content of the diet for groups 1, 2 and
8, including their young, was then increased to
0.05% after whelping. After 2} months the entire
mink colony was returned to the ranch and fed
the same diet without arsanilic acid. The adults
and young were observed 3 months later to be in
excellent health and with prime coats. Growth,
food consumption, breeding, whelping and rearing
of young was normal throughout. The number of
young born and raised in the laboratory colony
was equal to the average results experienced on a
well managed mink ranch. Mink show high toler-
ance to arsanilic acid, i.e. 10 times normal feeding
level, similar to chickens and rats. This degree of
tolerance is higher than that shown by dogs,
turkeys and swine.

FEDERATION PROCEEDINGS

Volume 18

1179. Conversion of D-erythrose-4-phosphate
plus phosphoenolypyruvate to intermedi-
ates in shikimic acid formation. P. R.
SRINIVASAN* AND Davin B. Sprinson. Dept. of
Biochemistry, Columbia Univ., New York City.
In a previous report (Srinivasan, P. R., M.

Karaatri AND D. B. Sprinson. J. Am. Chem. Soc.

77: 4943, 1955) it was shown that erythrose-4-phos-

phate (E-4-P) plus phosphoenolpyruvate (PEP)

are converted quantitatively by cell-free extracts
of EF. coli mutant 83-24 (blocked immediately after
shikimic acid (SA)) to SA or its precursor, 5-de-
hydroshikimate (DHS). The products of the first
reaction in this sequence were assumed to be in-
organic phosphate (Pj) and 2-keto-3-deoxy-7-
phospho-p-glucoheptonic acid (KDPH). The latter
compound has now been obtained by synthesis

(RotuscuHitp, J., M. Sprecuer, D. B. SprRINSON

AND J. E. WASHBURN. Unpublished results) and is

readily utilized for DHS formation. There is evi-

dence that KDPH is identical with .;compound

‘V,’ a phosphorylated a-keto acid accumulated by

E. coli mutant 83-3 (blocked before dehydroquinic

acid) and converted to DHS by extracts of strain

83-24 (Kaan, E. B. anp F. Lerrner, reported by

B. D. Davis. Federation Proc. 14: 691, 1955).

Fractionation of protamine-treated extracts of

strain 83-24 with (NH,)2SO,4 and with acetone

yielded 2 types of fractions. With one, PEP dis-
appeared only in the presence of E-4-P, DHS was
not formed, and KDPH was not utilized. With the
other, KDPH was converted to DHS. Incubation

of E-4-P plus PEP with extracts of strain 83-24

results in a very rapid release of 2 m of P; anda

much slower formation of DHS. With KDPH 1 m

of P; is similarly released. These results suggest

the formation from KDPH of a nonphosphorylated
acyclic intermediate in SA synthesis.

1180. Propionate oxidation by cell-free ex-
tracts of Clostridium propionicum. E. R.
SraptMaN. Natl. Heart Inst., Natl. Insts. of
Health, PHS, Bethesda, Md.

In the presence of ammonia and catalytic
amounts of acetyl-P, cell-free extracts of C.
propionicum catalyze the oxidation of 1-C14-propi-
onate to C'*-labeled B-alanine. The available evi-
dence indicates that the oxidation involves the
following reactions: Acetyl-P + propionate +
CoA — propionyl CoA + acetate + Pi... (1);
Propionyl CoA $5 acrylyl CoA + 2H... (9);
Acrylyl CoA + NH; — £B-analyl CoA... (8).
With crude dialyzed extracts, molecular oxygen or
tetrazolium dyes can serve as electron acceptors
for propionate oxidation. In reactions 2 and 3, the
acyl-CoA derivatives may be replaced by the cor-
responding acyl pantotheine analogues. The above
sequence of reactions is supported by the following
observations: a) no oxidation of free propionate

de
sy’
ac

fre
lys
rin
gel
the
bo
ag:

alb
the
ing

the
alb
syr
boc
pol
cou
gav
tati
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pre
pol,
phe
con
is 1

teri
anti

eX
i. R.
is. of

ily tic
of C.
yropi-
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s the
te +
. (1);
. (2);
. (8).
ren or
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e cor-
above
owing
onate

March 1956

occurs in the absence of added acetyl-P; b)
acetyl-P 4s not required for the oxidation of
propionyl-CoA (or propiony! pantotheine) ; c) the
reverse of reaction 2 has been measured directly
by showing that in the presence of reduced safra-
nine as an electron donor, acrylyl-pantotheine is
reduced to propiony! pantotheine; d) evidence for
reaction 3 was obtained by showing that in the
presence a partially purified enzyme preparation
and ammonium ions, acrylyl pantotheine dis-
appears (as measured by a decrease in optical
density at 263 ») and that B-alanyl pantotheine is
formed in stoichiometric amounts. The f-alanyl
pantotheine was identified by direct chemical,
chromatographic and spectrophotometric com-
parison with an authentic sample of #-alanyl
pantotheine. Attempts to demonstrate reversi-
bility of reaction (3) have not been successful.

1181. Antigenicity of synthetic polypeptides.
Mark A. STAHMANN, Hirosur Tsvuyvk1,* Karu
F. Wernke,* Cuaup LAPRESLE* AND PIERRE
GRABAR.* Dept. of Biochemistry, Univ. of Wiscon-
sin, Madison, and Service De Chemie Microbienne,
Institut Pasteur, Paris, France.

The antigenicity of synthetic polypeptides was
demonstrated by injecting into rabbits soluble
synthetic polypeptides prepared from glutamic
acid or lysine and also rabbit or bovine albumin
modified by coupling with polypeptides prepared
from leucine, phenylalanine, glutamic acid or
lysine. The immunsera were studied by means of
ring tests, by formation of specific precipitates in
gelified media and by immunoelectrophoresis of
the antigens and immunsera. Three types of anti-
bodies were demonstrated within immunsera
against bovine albumin coupled to the poly-
peptides: one with a specificity to the bovine
albumin, another to the polypeptide and one to
the albumin and polypeptide together. Precipitat-
ing sera from rabbit albumin coupled with poly-
peptides contained 2 types of antibodies, one to
the polypeptide, another to a portion of the rabbit
albumin and the polypeptide. Antisera from the
synthetic polypeptides contained only one anti-
body which did not precipitate the free synthetic
polypeptide but did precipitate bovine albumin
coupled to the corresponding polypeptide. It also
gave cross reactions with several proteins. Precipi-
tation was not inhibited with amino acids or
polypeptides. From the density and position of
precipitation lines, the most antigenically active
polypeptide was that from leucine, followed by
phenylalanine and then glutamic acid. Lysine was
considerably less active. These results show that it
is now possible to study with synthetic poly-
peptides of known chemical structure the charac-
teristic structural requirements necessary for
antigenicity and antigen-antibody reactions and

D ons bh

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

361

to foresee the theoretical possibility of preparing
synthetic vaccines from synthetic polypeptides.

1182. Effect of glucagon on renal excretion of
electrolytes. ALFRED STauB, VIVIEN SPRINGS
AND Haroup Exrick (introduced by Cosmo G.
MackKENZIE£). Radioisotope Service, VA Hosp.
and Dept. of Biochemistry, Univ. of Colorado
School of Medicine, Denver.

The fate and action of I'*!-labeled and crystal-
line glucagon were studied in normal dogs. A 5%
glucose solution was infused continuously into
anesthetized (Nembutal) animals in order to
produce diuresis. I'*!-glucagon was administered
intravenously after the urine flow reached 2
ml/min. Chromatographic analysis of blood and
urine showed that a considerable portion of the
protein bound I'*! (at least 30% in 90 min.) was
rapidly converted in the body to radioiodide. The
excretion of inorganic I'*! in the urine following
the injection of I'*!-glucagon was 3 or 4 times
greater than when an equivalent amount of [!#!
was injected alone, suggesting that glucagon had
accelerated the excretion of the liberated iodide.
This hypothesis was verified when purified
glucagon was shown to increase markedly the
excretion of radioactive iodide administered as
I'3!, Moreover, this action of glucagon was not
limited to I-. It was equally effective in the case
of Cl-. Thus, a single dose of glucagon increased
the excretion rate of Cl- from a base level of 25
mg/hr. (expressed as NaCl) to over 400 mg/hr. The
excretion of Nat, K+, and PO,-—~ was likewise
enhanced. Glucagon did not increase the glomeru-
lar filtration rate or the blood chloride level. This
suggests that its action is on the kidney tubules.

1183. Tumor-host relationships in formate
metabolism: oxidation of formate by mouse
liver homogenates. ABRAHAM M. STEIN* AND
Joun W. Ment. Dept. of Biochemistry and Nu-
trition, Univ. of Southern California, Los Angeles.
We have endeavored to extend the observation

of in vivo inhibition of formic acid oxidation by

tumor (Federation Proc. 14: 286, 1955) to mouse
liver homogenates. Preliminary observation
showed that oxidation of formate-C' was stimu-
lated by xanthine, hypoxanthine and acetaldehyde
in the course of 1 hr. incubation. In dialyzed liver
homogenates supplemented with acetaldehyde,
the reaction rate follows Lineweaver-Burk kinetics
with respect to acetaldehyde concentration:
Vmax = 10.6 um C%O2/gm liver/hr.; Km = 0.006 m
acetaldehyde. The latter value is in good agree-
ment with the value of 0.007 m found by Gordon
et al. (Biochem. J. 37: 764, 1940) for pig liver ‘alde-
hyde oxidase.’ Inosine stimulates the oxidation of
formate-C™ in dialyzed homogenates; at high
concentration of inosine, acetaldehyde is ineffec-

362

tive in further stimulating the reaction, suggesting
that formate oxidation is limiting in this situation.
In agreement with oxygen uptake values obtained
with hypoxanthine, increasing the inosine con-
centration prolonged the initial rate of oxidation
without affecting the maximum rate of reaction.
In the range 0.01-0.10 m formate, maximal rate
of oxidation was obtained with 0.002 m inosine in
1 hr. incubation. At this concentration the oxida-
tion of formate follows Lineweaver-Burk kinetics:
Vmax = 28.4 um formate/gm liver/hr.; Km = 0.0145
m formate. The application of this assay to livers
of normal and tumor-bearing mice has been
studied. (Supported by a grant-in-aid from the
Natl. Cancer Inst., Natl. Insts. of Health, PHS
(C-2340), and by a grant from the American
Cancer Society.)

1184. Inhibition of protein degradation by
amino acid analogues. D. STEINBERG AND
M. VauGHAN (introduced by Fioyp 8. Dart).
Natl. Heart Inst., Natl. Insts. of Health, PHS,
Bethesda, Md.

Rat liver and kidney proteins were labeled by
injecting C'*-amino acids intraperitoneally 3
days prior to killing. Trichloroacetic acid-soluble
‘amino acid’ radioactivity (material adsorbed on
Dowex 50-H*+ and eluted by 2 N NH,OH) was
determined before and after 1-4 hr. incubation of
slices at 37°C in pu 7.4 Krebs-Ringer phosphate.
Release of radioactive ‘amino acids’ continued,
although at a declining rate, for 4 hr. As reported
by Simpson (J. Biol. Chem. 201: 148, 1953) this
release was strongly inhibited by anaerobiosis
and by 10-* m dinitrophenol (DNP). In searching
for more specific inhibitors it was found that
o- and p-fluorophenylalanine also inhibited ap-
proximately 40% at 2 X 107? m. Similar results
were obtained using labeled phenylalanine,
alanine or lysine. Because, as in Simpson’s studies,
unlabeled carrier was added to ‘trap’ the radio-
active ‘amino acids’ released, these isotopic results
might reflect only variations in effectiveness of
the penetration of carrier into cells. Net break-
down (increment in trichloroacetic acid-soluble
nitrogen (NPN)) was therefore studied under
identical conditions. In the average 4-hr. incuba-
tion NPN corresponding to about 3% of the slice
protein nitrogen was released. This net NPN
release was strongly inhibited by anaerobiosis,
by DNP and by o-fluorophenylalanine. On the
other hand, autolysis of liver homogenates at pH
5.0 was not significantly inhibited. The results
support the hypothesis that rat liver proteins are
in true dynamic state. ‘Physiological,’ intracellu-
lar protein degradation appears to be an organized
process more complex than direct catheptic
hydrolysis.

FEDERATION PROCEEDINGS

Volume 15

1185. Utilization of methionine for synthesis
of choline in rats and tumor-bearing mice
studied in vivo or in vitro. JAKkosB A. STEKOL,
SripnEy Weiss,* Eruent I. ANDERSON* AND
Auice WatsEeNn.* Lankenau Hosp. Research Inst,
and Inst. for Cancer Research, Philadelphia, Pa.
We previously reported that the lowered utiliza-

tion of the methy] of methionine for choline forma-

tion in intact folic acid-deficient rats can be
restored to normal by supplementation with
dimethylaminoethanol or citrovorum factor, but
not with aminoethanol or monomethylamino-
ethanol. Similar observations have now been
recorded by employing liver slices of folic acid-
deficient rats. These results suggest that the
transfer of the methyl of methionine to choline
does not involve folic acid derivative and that it
is substrate specific. This substrate is dimethyl-
aminoethanol, and a folic acid derivative appears
to be involved in a de novo synthesis of its methy]
groups. Compared to the livers of tumor-bearing
mice, the tumors (S* and Krebs 2) showed poor
ability to utilize methyl of methionine for choline
formation in vivo or in vitro. The extent of in-
corporation of choline-CH;-C' was greater in
tumors than in livers. The tumors utilized radio-
formate or serine-3-C'* for methionine synthesis,
but poorly for choline formation. Neither ATP,
citrovorum factor nor aminoethanol, singly or
together, improved choline synthesis by tumors.
Dimethylaminoethanol supplementation im-
proved utilization of methyl of methionine for
choline formation by tumors in vivo or in vitro. It
appears that in the tumors employed, the enzyme
or enzymes involved in the formation of dimethyl-
aminoethanol is the limiting factor in choline
synthesis from methyl] of methionine. Further de-
tailed studies of this possibility are in progress.

(Aided by grants from Natl. Cancer Inst., Natl.

Insts. of Health, PHS, and from the Atomic

Energy Commission.)

1186. Incorporation of C'‘-amino acids into
proteins of leaf disks and cell-free fractions
of tobacco leaves. Mary L. STEPHENSON* AND
Pau C. ZamMeEcNIK. Huntington Memorial Hosp.
of Harvard Univ., Massachusetts General Hosp.,
and Dept. of Biological Chemistry, Harvard Med.
School, Boston.

When tobacco leaf disks were incubated with
0.04 m C'4-carboxy] labeled t-valine, p1i-leucine,
pL-alanine or glycine, the amino acid was in-
corporated into protein at approximately 0.6%/hr.
for 5 hr. When disks were incubated 1 hr. and then
cell fractions isolated by homogenization and
centrifugation, all fractions were equally labeled.
As in other in vivo isotopic experiments using
N'“H,Cl and CO. (MENEGHINI, COMMONER,

fre
in
ins
4
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ple
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esis
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KOL,
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oline
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adio-
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March 1956

JEENER) virus becomes labeled. Leaf disks which
had been infected with tobacco mosaic virus (gift
of B. Commoner) 10 days previously were incu-
bated for 2 hr. with u-valine-1-C' and fraction-
ated; the specific activity of the virus recovered
was 20-60% of the other fractions. Cell-free sys-
tems incorporated both C'‘-valine and C'*-leucine.
Leaves were minced in 0.5 m glucose, 0.02 m K
phosphate buffer at pH 7, and 0.01 m MgCl, and
a 700 X g supernatant was incubated with labeled
amino acid at 20°C. in 95% O2-5% COz, and then
fractionated. In contrast with bean hypocotyl
homogenates (G. C. WeBsTER) and rat liver homo-
genates, the microsome protein (105,000 XK g
pellet) was no more highly labeled than any other
fraction. Finally, when isolated fractions were
incubated separately, the microsome fraction was
inactive, whereas the 15,000 X g pellet and 105,000
X g supernatant both incorporated amino acid
(at 1%/hr.) for 15 min. Incorporation into chloro-
plasts (1,000 X g pellet), although no more rapid,
continued for 1 hr., and was stimulated 2-3-fold
by oxygen and light. The chloroplasts apparently
are unique in having necessary factors which allow
incorporation to continue after other isolated
fractions have ceased functioning.

1187. Estimation of alpha-ketolic steroids by
reduction of ferricyanide. Norman R.
STEPHENSON (introduced by L. I. Pugsley).
Food and Drug Labs., Dept. Natl. Health and
Welfare, Ottawa, Canada.

A procedure based on a modification of Folin’s
micromethod for blood sugar (J. Biol. Chem. 77:
421, 1928), has been developed for the estimation
of small quantities of alpha-ketolic steroids. The
ferrocyanide produced was measured as Prussian
blue in an Evelyn photoelectric colorimeter. With
the 620 filter, a straight line was obtained by
plotting the density (2-Log G@) of the chromogen
against the concentration of the reducing steroid
over the range from 0 to 40 ug. The ability of an
adrenal corticoid to reduce ferricyanide in an
alkaline solution was related inversely to the
number of polar groups in the molecular structure.
This was evident when the steroids were arranged
in descending order according to their reducing
activity : desoxycorticosterone, corticosterone and
dehydrocorticosterone, cortisone and cortisol,
desoxycortisol, prednisone and _ prednisolone,
tetrahydrocortisone. Steroid compounds without
the alpha-ketolic grouping, such as adrenosterone,
progesterone, pregnenolone, testosterone and
methyltestosterone displayed only slight reducing
activity. Apparently a ketone at Cs; accompanied
by a double bond in the 4,5 position had very
little effect on the alkaline ferricyanide. The
acetate esters of the corticosteroids had the same

S aon bk

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

363

reducing power, on an equimolar basis, as the
free alcohols. The method has been used for the
quantitative estimation of adrenal corticoids in
pharmaceutical preparations.

1188. Characterization of myosin from nor-
mal and failing dog heart. Howarp STERN,*
Eric ELLENBOGEN* AND Ropert E. OLson.
Dept. of Biochemistry and Nutrition, Grad. School
of Public Health, Univ. of Pittsburgh, Pitts-
burgh, Pa.

Chronic congestive heart failure, characterized
by edema, ascites, weakness, reduced exercise
tolerance, cardio- and hepatomegaly and elevated
end-diastolic filling pressures, has been produced
in dogs by surgical avulsion of the tricuspid valve
and stenosis of the pulmonary artery. Myosin
was isolated from the hearts of these animals and
from normal controls, as was previously described
(Federation Proc. 14: 207, 1955) and its physico-
chemical properties studied. The following prop-
erties were found for myosin from normal dog
heart: sedimentation constant (c = 0) was 5.948,
dS/de (c in gm/100 ml) was —3.0, partial specific
volume 0.74, boundary spreading coefficient 2.3 X
10-7 em?2/sec., intrinsic viscosity 0.85, and ATP-ase
activity (Qp) 150. From these constants, the
molecular weight of normal cardiac myosin was
estimated to be about 240,000 and the axial ratio
about 30-1. In the failing heart, on the other
hand, the following properties were found: sedi-
mentation constant (c = 0) was 6.45 8, dS/de
(c in gm/100 ml) was —5.2, partial specific volume
0.71, boundary spreading coefficient 0.85 x 1077
cem?/sec., intrinsic viscosity 2.05, ATP-ase activity
(Qr) 152. From these constants, the molecular
weight of cardiac myosin in the failing heart was
estimated to be about 740,000 and the axial ratio
about 80-1. The possibility that these changes in
the molecular configuration of myosin in cardiac
failure are related to the changes in contractility
observed in the failing heart is under intensive
study. (Supported in part by the American Heart
Association and Public Health Service.)

1189. Acetoacetyl glutathione thioesterase
and a mechanism of deacylation of aceto-
acetyl CoA. JosepH R. STERN AND GEORGE
I. Drummonp.* Dept. of Pharmacology, Western
Reserve Univ., Cleveland, Ohio.

Soluble extracts of animal tissues and yeast
have been found to contain a specific thioesterase
which catalyzes the reaction (1) acetoacetyl-S-
glutathione — acetoacetate + glutathione. This
enzyme has been partly purified from ox liver
and shown to be distinct from previously de-
scribed thioesterases for acetyl, crotonyl and
lactoyl glutathiones. Neither the crude liver

364

extract nor the purified enzyme hydrolyze
acetoacetic thioesters of CoA, pantetheine, N-
acetyl-8-mercaptoethylamine, cysteine or mer-
captoacetic acid. However, the deacylation of
acetoacetyl-S-CoA and acetoacetyl-S-pantetheine
proceeds in the presence of ox liver fractions
(treated with iodoacetamide to inhibit thiolase),
provided reduced glutathione (GSH) is added.
This has been shown to be the result of coupling
reaction (2) acetoacetyl-S-CoA (acetoacetyl-S-
pantetheine) + GSH = acetoacetyl-SG +
CoA-SH (pantetheine) with reaction (1) to effect
the net reaction (3) acetoacetyl-S-CoA (aceto-
acetyl-S-pantetheine) — acetoacetate + CoA-SH
(pantetheine). Over a considerable range of ex-
perimental conditions, reaction (2) is non-
enzymatic and lacks thiol and thioester specificity.
Thus far, it has not been possible to demonstrate
unequivocally an enzyme catalyzing it. The
coupling of reactions (1) and (2) constitutes a
mechanism for deacylation of acetoacetyl-S-CoA
and acetoacetyl-S-pantetheine, which may con-
ceivably operate in intact liver. These ox liver
fractions also catalyze the over-all synthesis of
acetoacetate from acetyl-S-CoA. The properties
of the nonenzymatic transacetoacetylation reac-
tion and of acetoacetyl glutathione thioesterase
will be presented and discussed in relation to
those of the over-all acetoacetate synthesizing
system.

1190. Relation of molecular weight to
metabolic activity of glycogen in liver and
muscle. MarsorigE R. Stetren anp DeWi1TT
Stetren, Jr. Natl. Inst. of Arthritis and
Metabolic Diseases, Natl. Insts., of Health,
Bethesda, Md.

Glucose-C™ has been administered to animals
and glycogen has been isolated from liver and
muscle 3-6 hr. thereafter. Because it was found
that treatment of glycogen with hot concentrated
alkali, as is done in the classical isolation method,
resulted in a product of greatly diminished molec-
ular weight, the samples studied were isolated
by cold trichloroacetic acid extraction. Purified
glycogen samples were fractionated by differential
centrifugation or by fractional precipitation with
ethanol. By either procedure, the fractions se-
cured initially contained molecules of higher molec-
ular weight whereas the later fractions contained
material of progressively lower molecular weights,
as determined by the method of light scattering.
When muscle glycogen was subjected to such
fractionation, the highest specific radioactivity
was always found to be associated with those
fractions of highest weight average molecular
weight, with progressive decline in specific ac-
tivity as the molecular weight of succeeding frac-
tions diminished. With liver glycogen, the highest

FEDERATION PROCEEDINGS

Volume 15

specific activities were consistently associated
with fractions of the lowest molecular weight, with
aclear trend toward a negative correlation between
specific activity and molecular weight. The meta-
bolic inhomogeneity of glycogen, previously
established with respect to participation of the
several tiers of the glycogen molecule, may now
be extended to include variations in reactivity of
glycogen molecules of differing sizes in the poly-
disperse population.

1191. Valine metabolism and penicillin bio-
synthesis. Cart M. Stevens, CuHester W.
DeLone,* Pran VowHRA* AND EpwarpD
InAMINE.* Fulmer Chemical Lab., State College
of Washington, Pullman.

The reports of Arnstein et al. (cf. Biochem. J.
60: xxxiv, 1955) and work from this laboratory
(ef. J. Biol. Chem. 211: 297, 1954) have established
that L-cystine and pu-valine are utilized as pre-
cursors of the penicillin molecule. The entire
molecule of cystine and the carbon skeleton of
valine are incorporated, while the retention of
valine nitrogen is uncertain. A logical combina-
tion of these 2 precursors, N,N’-(L-cystyl)di-p-
valine was tested, but in short-term competitive
utilization experiments it was much inferior to
L-cystine. A comparison of the rates of utilization
of added L- and p-valine showed that, whereas
L-valine is rapidly incorporated, there is a con-
siderable lag before maximal incorporation of
p-valine is reached. The conversion of D-valine
to u-valine under these conditions was demon-
strated by analysis of mycelial hydrolysates. From
cultures to which p-valine-1-C'* had been added,
the isolated L-isomer contained more than 90%
of the total radioactivity of the mycelial valine.
Added t-valine is utilized for penicillin biosynthe-
sis in preference to alpha-ketoisovaleric acid,
pL-alpha, beta-dihydroxyisovaleric acid, beta-
hydroxy-pt-valine, or beta, beta-dimethylacrylic
acid. Further evidence for a close relationship
between the biosynthesis of valine and of peni-
cillin is indicated by the fact that addition of ace-
tate-1-C™ results in the production of mycelial
valine and of penicillamine (from penicillin) with
similar labeling, and that the incorporation of
labeled acetate into penicillamine is reduced by
addition of nonlabeled t-valine to the medium.

1192. Metabolism of estrone-1l6, estradiol-
3,16a and estradiol-3,168 in men. BENJAMIN
F. StimmMe.. Rees-Stealy Clinic Research Fndn.,
San Diego, Calif.

The synthetic 3, 16-estradiols, unlike estrone-16,
are quite chromogenic in the Kober test. Like
estradiol-3,17a they require no heating to develop
the yellow color characteristic of the first stage of
the Kober test. They may be distinguished spec-

eR I A en es ila sie ite dale ii a eos Ma fh ek

me 15

iated
, with
tween
meta-
ously
f the
7 now
ity of
poly-

| bio-
R W.
yWARD
Jollege

em. J.
ratory
lished
is pre-
entire
ton of
ion of
nbina-
1)di-p-
etitive
rior to
ization
hereas
a con-
ion of
-valine
lemon-
;. From
added,
in 90%
valine.
synthe-
s acid,
beta-
lacrylic
ionship
f peni-
of ace-
ry celial
n) with
tion of
iced by
dium.

radiol-
INJAMIN
, Fndn.,

rone-16,
3t. Like
develop
stage of
ed spec-

March 1956

trophotometrically from estradiol-3,17a by their
failure to’develop color in the dilute iron-Kober
(Harnni, E. O. J. Amer. Pharmaceut. Assoc. 39:
1950) and formic acid (Boscorr, R. J. Nature
164: 140, 1949) tests. The 3,16-diols yielded similar
absorption spectra (320-500 ») with a) 90% sul-
furic, b) 15% fuming sulfuric and c) 100% phos-
phoric acids. Hence we were unable to distinguish
between them _ spectrophotometrically. Men
treated with 10 mg of estrone-16 excreted in the
urine an alcoholic, nonketonic Kober chromo-
gen(s) which was characterized spectrophoto-
metrically as estradiol-3,16 (10-20% yield). Only
a trace of unchanged estrone-16 (except for con-
jugation) was detected in the urine. No C-17 sub-
stituted estrogens such as estrone, estradiol and
estriol were detected above pretreatment levels.
Men treated with a) estradiol-3,168 and b)
estradiol-3,16a excreted in the urine the Kober
chromogen(s) which was. characterized as
estradiol-3,16 (15-30% yield). The 8-epimer, but
not the a-epimer yielded a small amount (approx.
6%) of estrone-16. On the other hand, men treated
similarly with the natural estrogen, estradiol-
3,178, excreted principally estrone, estriol and
only a trace of unchanged estradiol-3,178 in the
urine. Location of the ketone group of estrone
at C-16 instead of C-17 enhances reduction to the
corresponding carbinol and hinders C-16, C-17
glycol formation in men. (Aided by Public Health
Service Grant C759 C6 and American Cancer
Society EDC-10A.)

1193. Isolation of DNP-peptides from DNP-
polyvalyl-proteins. ALFRED StRacHER, W. H.
KonIGSBERG AND R. R. Brecker (introduced by
M. L. CaLpwE tL). Dept. of Chemistry, Columbia
Univ., New York City.
Dinitrophenyl-polyvalyl-proteins, prepared by

the reaction of N-carboxy-pu-valine anhydride

with the protein and subsequent treatment with
dinitrofluorobenzene, yielded DNP-peptides after
hydrolysis in 5.7 N HCl at 105°C for 16 hr., the
conditions usually employed for the isolation of
DNP-amino acids. The peptides were separated on
silicic acid columns using water-saturated chloro-
form and butanol-chloroform mixtures as the
eluting solvents. The purity of the peptides was
further checked by 2-dimensional paper chroma-
tography and paper electrophoresis. Peptides
have been isolated in this way from chymotrypsin,
trypsin, lysozyme, egg albumin and insulin. Under
identical experimental conditions, no DNP-
peptides were found when the DNP derivatives
of unmodified, poly-t-phenylalanyl-, or poly-
glycyl-proteins were hydrolyzed. When insulin
was used as a model protein, a peptide containing
the following amino acids in addition to the added
valine was isolated in about 50% yield: Ala (C-

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

365

terminal), Lys, Pro, Thr, Arg, Tyr, Phe, Glu,
and Gly (N-terminal). This peptide would be
expected from the C-terminal end of the phenyl-
alanine chain, and contains the only lysine and
proline in the molecule, The stability to acid
hydrolysis of peptides containing valine is well
known and has been attributed to steric hindrance.
The manner in which the valine peptides provide
stabilization of peptide bonds surrounding lysine,
the point of attachment of the valine peptides thus
far isolated, might well involve other factors.

1194. Isotope tracer studies on mechanism
of lysine biosynthesis in Escherichia coli.
Murray SrrassMan, AuicE J. THOMAS AND
Sripney Weinuouse. Lankenau Hosp. Research
Inst. and Inst. for Cancer Research, Phila-
delphia, Pa.

Data of Mitchell and Houlahan with mutant
strains of Neurospora crassa showed that a-amino-
adipic acid is a precursor of lysine in this organism.
Isotope tracer studies indicated that a-amino-
adipic acid may also be a precursor of lysine in
T. utilis, and a mechanism for its formation was
suggested, involving a variant of the citric acid
cycle in which a-ketoglutarate, rather than oxal-
acetate participates in the initial condensation,
Nutritional studies with EF. coli mutants point to
a different synthetic mechanism in this organism,
in which a,e-diaminopimelic acid is a lysine
precursor. To obtain further information con-
cerning the routes of lysine and diaminopimelic
acid synthesis in E. coli, a wild strain of this bac-
terium was grown on glucose as the principal
carbon source, together with tracer quantities of
acetate-1- and 2-C"4 and lactate-3-C''. The lysines
were isolated from the cell protein and degraded
chemically to determine the distribution of C™.~
The following distribution patterns were observed
in the lysines, all three of which were highly
labeled.

H:NCH:—CH:—CH:—CH:—CHNH:—COOH
0 63 0 0 36

CH:C“OOH

C“uH;COOH 22 6 32 32 7

C“H;CHOH 11 31 38 15 5
COOH ‘

Comparison of these with previous results indi-
cates that the mechanism followed in 7. utilis
does not occur in E. coli. If so, activity would have
been observed in carbon 6 of lysine formed from
CH;C“OOH.

1195. Studies in vitro of mineralization proc-
ess. Bastt Srrates* anp W. F. Neuman.
Dept. of Radiation Biology, Univ. of Rochester,
Rochester, N. Y.

A direct approach was taken to the problem of
formation of hydroxy apatite, (the prototype
mineral of bone) from neutral solutions at physi-
ological ionic strength. Solutions containing

366

varying concentrations of calcium and phosphate
were mixed and equilibrated for 10 days at 25°C.
The supernates of the mixtures were clarified
either by high speed centrifugation or filtration
through ‘molecular’ filters (millipore paper) and
analyzed. Precipitates formed only when the prod-
uct aca++-anpo,- = 540.3 X 1077 was exceeded.
This product is in good agreement with the pub-
lished K,, of CaHPO,-2 H,O. At pu 6.2, the solid
phase was indeed crystalline CaHPO,-2 H.0 as
shown by analysis, physical properties and x-ray
diffraction. At the higher pu of 6.9 and 7.4, the
initial precipitate exhibited a molar ratio, Ca/P,
of unity, but this ratio rapidly increased and, at
10 days, the solid phase had converted to hydroxy
apatite as shown by analysis and x-ray diffraction.
Next an attempt was made to demonstrate forma-
tion of hydroxy apatite by ‘nucleation’ from
undersaturated solutions. Fibrin and gelatin failed
to initiate precipitation, but hydroxy apatite
crystals and reconstituted collagen fibers (pre-
pared according to Schmitt and co-workers)
did result in crystal formation from otherwise
stable solutions. (Based on work performed under
contract with the Atomic Energy Commission at
the Univ. of Rochester Atomic Energy Project,
Rochester, N. Y.)

1196. Structure and function of mitochondria
in irreversible hemorrhagic shock. J. G.
Srrawitz AND H. Hirt (introduced by W. H.
McSuHan). Univ. of Wisconsin Med. School,
Madison.

Dogs were subjected to irreversible hemorrhagic
shock by the Wiggers procedure. Portions of their
heart, liver and kidney ground in formaldehyde
and examined under a phase contrast microscope
revealed mitochondria which differed in size and
shape from those prepared from paired control
animals. Heart mitochondria isolated by differ-
ential centrifugation from ‘shock’ dogs tended to
be smaller in size than normal controls. Their
capacity to oxidize Krebs cycle substrates was
unimpaired but their P/O ratios tended to be
lowered.

1197. Seattered fatty acid peroxidase. P. K.
Stumpr. Dept. of Plant Biochemistry, Univ. of
California, Berkeley.

Previous studies (CASTELFRANCO, STUMPF AND
Contopoutous. J. Biol. Chem. 214: 507, 1955)
indicated that a protein system obtained from
extracts of germinating peanut cotyledons
catalyzed the oxidation of palmitic acid-1-C™ to
CO, and unknown cleavage products. Essential
for activity was the presence of an L-a-hydroxy-
monocarboxylic acid or of glycolic acid. Experi-
ments now reveal that the function of hydroxy
acids was to serve as a substrate for glycolic

FEDERATION PROCEEDINGS

Volume 16

oxidase which occurs in low concentrations in the
peanut protein system. One of the reaction
products of glycolic oxidase activity is H2O2. The
following evidence supports the contention that
in peanut extracts a specific fatty acid peroxidase
occurs which in the presence of H2O: oxidatively
decarboxylates long chain saturated fatty acids.
When reagent H:O2 is added to the reaction sys-
tem, no reaction is observed; however, the follow-
ing systems have proven equally effective in
generating H.O2 for the peroxidase system: 1)
glycolic acid-glycolic oxidase, 2) glucose-glucose
oxidase, 3) L-leucin-L-amino acid oxidase and 4)
glucose-methylene blue, O2, DPN and glucose
dehydrogenase. Catalase does not inhibit the
oxidative systems; however CN, azide, and
imidazole are effective inhibitors. The fatty acid
peroxidase will catalyze the release of C'*O2 from
palmitic-1-C™ but not from palmitic-2-C™, -3-C™,
-11-C', and -15-C'. Steric-1-C™, palmitic-1-C",
myristic-1-C'™ are reactive. Lauric-1-C™ and the
lower homologs are inert. Horse radish peroxidase
does not replace the peanut protein system. Evi-
dence is presented suggesting the formation of
long chain fatty aldehydes as one of the reaction
products. (Supported in part by a research grant
from the Natl. Science Fndn.)

1198. Multiple function of lactic oxidative
decarboxylase from Mycobacterium phlei.
W. B. Sutton (introduced by G. H. A. Ctowgs).
Lilly Research Labs., Eli Lilly and Co., Indian-
apolis, Ind.

Earlier phases of the present investigation con-
cerning the lactic oxidative decarboxylase from
Mycobacterium phlei have established that for
each mole of lactate metabolized by this enzyme,
1 m of oxygen is utilized and 1 M each of acetate
and CO, is formed. The flavoprotein character of
the enzyme (riboflavin-5’-phosphate), as well as
the absence of associated coenzymes and iron
porphyrin compounds including cytochrome bz,
has been established (Surron. J. Biol. Chem. 216:
749, 1955). Certain atypical aspects of the enzyme
activity, including the failure to produce H:0;
and the absence of a keto acid intermediate in the
oxidation of lactate, have been recognized. The
dual function of the enzyme, i.e. oxidation and
decarboxylation of lactate, as associated with a
single protein unit, conflicts with the accepted
views concerning enzyme function. This activity
is, however, reminiscent of the malic enzyme of
Ochoa et al., which catalyzes the oxidative de-
carboxylation of malate to pyruvate and CQ:
without intermediary formation of oxalacetate.
The absence of H2O2 has been established by
several means including the addition of catalase
to the reaction mixture and the failure to observe
coupled oxidation of alcohol. The addition of

—pae: a a a a ee a

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92
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March 1956

carbonyl fixatives to the reaction mixture does not
interfere with oxygen utilization or the production
of COz. This latter result clearly demonstrates
that a free carbonyl compound is not the inter-
mediate product resulting from the oxidation of
lactate. The experimental results obtained suggest
that some form of peroxidation of the lactate oxi-
dation product occurs at the enzyme surface,
followed by evolution of COz and the formation
of acetate.

1199. Effects of arsenite and azide on phos-
phate-turnover reactions in mitochondria.
MarsorrE Swanson. Dept. of Biochemistry,
Bowman Gray School of Medicine, Winston-
Salem, N. C.

The effects of arsenite and azide on the mito-
chondrial phosphate-turnover reactions (ATP-
splitting, ATP-inorganic phosphate exchange and
oxidative phosphorylation) have been studied
under a wide variety of conditions. The effects of
arsenite are widely variable. Part of the vari-
ability appears to be related to small changes in
the ionic concentration of the medium. Arsenite
has little effect in slightly hypotonic media, but
evokes ATP-splitting activity and markedly
inhibits both exchange and phosphorylation in
slightly hypertonic media. Certain substances
such as versene, succinate and some of the buffers
used appear to afford a considerable degree of
protection against arsenite. The effects of azide
are sharply diphasic. At low concentrations, azide
definitely inhibits ATP-splitting but affects the
other reactions hardly at all. Over a narrow range
of concentrations it evokes high ATP-splitting
activity, at still higher concentrations it again
inhibits ATP-splitting. In the higher concentra-
tion ranges, azide markedly inhibits both exchange
and phosphorylation, the degree of inhibition at-
tained depending on the nature of the oxidizable
substrate present.

1200. Incorporation of molecular oxygen into
ll 8-position of corticosteroids. Max L.
Sweat, R. A. Aupricu,* C. H. pe Bruin,*
W. L. Fowxks,* L. R. Hersert* ann H. §S.
Mason. Dept. of Physiology, Western Reserve
Univ. School of Medicine, Cleveland, Ohio, and
Univ. of Oregon Med. School, Portland.

Beef adrenal mitochondria convert desoxycorti-
costerone to corticosterone by the incorporation
of 1 atom of oxygen into the 8-position of carbon
atom 11. Experiments in which desoxycorti-
costerone was incubated in the presence of mito-
chondria, fumarate, magnesium, TPN and
molecular oxygen demonstrated an average of
92% theoretical incorporation of O2'. It is con-
cluded from these data that the oxygen 11 of the
ll-oxysteroids is derived from molecular oxygen.

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

367

The 11-8-oxidation of desoxycorticosterone is
dependent upon the presence of reduced triphos-
phopyridine nucleotide. Intact mitochondria
contain multiple enzyme systems which are
capable of generating TPNH from TPN and
Krebs cycle metabolites. In experiments with
soluble preparations, TPNH is added or is gen-
erated by adding TPN, glucose-6-phosphate and
glucose-6-phosphate dehydrogenase to the incuba-
tion media. The path for 11-8-oxidation does not
appear to involve cytochrome oxidase since cyto-
chrome oxidase is highly sensitive to cyanide
poisoning and is destroyed by acetone drying.
The slight inhibition of the 11-8-oxidase reaction
by cyanide is suggestive of a catalase-like enzyme.
The strong inhibition by diethyldithiocarbamate
suggests that the enzymic activity is associated
with a copper complex. (Aided by grants from
the Public Health Service.)

1201. Biosynthesis of spermidine and
spermine from putrescine. HERBERT TABoR,
Sanrorp M. RoseNTHAL AND CELIA WHITE
Tasor.* Natl. Insts. of Health, PHS, Be-
thesda, Md.

Spermidine [NH:2(CH2);NH(CH:2),NH»] and
spermine [NH2(CH:)sNH(CH:2),NH(CH:);NH)]
are present in high concentrations in various
tissues and microorganisms (J. Pharmacol. v. 116).
The present experiments with labeled putrescine
(N4H.C“H2CH2CH2C“H2NH:2) indicate that
putrescine is utilized as a unit in the biosynthesis
of these polyamines in growing cultures of Escher-
tchia coli and Aspergillus nidulans pu, (a putres-
cine-requiring mutant; Sneatu, Nature 175: 818
1955). #. coli was grown on an ammonia-glucose-
salt medium plus 33.7 um of labeled putrescine; the
washed cells (5.5 gm, wet) contained 11.3 um of
spermidine with 13.5 and 14.9% of the added C™%
and N!5, 28.4 um of putrescine with 10.3 and 11.2%
of the added C'* and N!, and traces of spermine.
The isotope dilution in the isolated compounds
demonstrates additional synthesis of putrescine
from unlabeled substrates. With A. nidulans 12.4
uM of putrescine were added to the medium; the
mycelia (15.5 gm, wet) contained 2.51 um of
spermidine with 14.4 and 16.7% of the added C4
and N#, and 5.97 um of spermine with 52.4 and
52.6% of the added C' and N*®. No significant
dilution of the isotope occurred in this putrescine-
requiring strain. Smaller incorporation into the
polyamines was observed (chiefly in spermidine)
when minced rat prostate was incubated with
C'4N putrescine (0.69% of the C' and 0.75%
of the N"5); no incorporation was observed with
minced liver, muscle, spleen and kidney. In all
the experiments the ratio of C!4:N* in the poly-
amines was essentially the same as in the added
putrescine.

368

1202. Bacterial riboside hydrolase. Y. TAKAGI
(introduced by E. O. Kerues). Natl. Insts. of
Health, PHS, Bethesda, Md.

Extracts of Lactobacillus delbriickii have been
shown by Kalckar (Pub. Staz. Zool. Napoli, 23:
Suppl. 87, 1951) to hydrolyze inosine. This enzyme
has now been purified about 50-fold by a procedure
including protamine precipitation, ammonium
sulfate fractionation and adsorption on calcium
phosphate gel. The activity was determined by
measuring the formation of uric acid in the pres-
ence of xanthine oxidase with inosine as the sub-
strate. With other substrates activity was
measured spectrophotometrically or by the rate
of appearance of reducing sugar. The final prepara-
tions are stable in the frozen state, although less
pure preparations lose appreciable activity in
24 hr. Inosine, adenosine and guanosine at 2.5 X
10-* m are hydrolyzed at nearly equal rates, while
cytidine and uridine at the same concentration
are hydrolyzed, respectively, at 3 and #5 the rate
with inosine. Imidazole acetic acid riboside is
hydrolyzed at the same rate as uridine. No change
in the relative activity with various substances is
observed during purification, suggesting that these
are hydrolyzed by the same enzyme in the ex-
tracts. No phosphate or Mg** requirement could
be detected. All of the 6-ribofuranosides tested
were hydrolyzed; a pyranoside (purine £-ribo-
pyranoside) was not attacked.

1203. Enzymatic isomerization of £,y-un-
saturated 3-ketosteroids. PauL TALALAY
(introduced by E. S. Guzman Barron).
Ben May Lab. for Cancer Research and Dept. of
Biochemistry, Univ. of Chicago, Chicago, Ill.
A‘-3-Ketosteroids are the products of chemical

(OPPENAUER) or enzymatic oxidations of A®-3-

hydroxysteroids. The enzymatic reaction can be

shown to occur in two steps: I, a DPN?*-linked

oxidation of the 3-hydroxyl group by a- or B-

hydroxysteroid dehydrogenases (J. Biol. Chem.

205: 823, 1953; 218: 661 and 675, 1956), followed by

II, isomerization of the double bond from the A®

to the A‘ position. Evidence has been obtained for

the enzymatic nature of reaction II, and that it is
catalyzed by an isomerase which is distinct from
the oxidizing enzymes of reaction I. The isomerase
is adaptive in Pseudomonas testosteroni and has
been assayed spectrophotometrically by the

conversion of 5-androstene-3,17-dione to 4-

androstene-3,17-dione (Amax = 248 my in H.0).

The bacterial enzyme has been purified to a turn-

over of 1300 um substrate/mg protein/min. at

pH 7.0 and 25°. This enzyme will also rearrange
5-pregnene-3,20-dione and 45(10)-estren-178-ol-
3-one to the corresponding a,§-unsaturated
ketones. Isomerase is also found in the soluble
portion of rat liver homogenates and in human

FEDERATION PROCEEDINGS

Volume 16

blood serum. Acid or base catalyzed isomeriza-
tions of 5-androstene-3,17-dione in media con-
taining D.O or T.0 result in the incorporation of
the isotopes into the product, whereas the
enzymatic isomerization at px 7.0 in such media
results in negligible pickup of the isotopes. The
enzymatic reaction probably involves a direct
transfer of hydrogen from position 4 to 6 on the
steroid skeleton.

1204. Preparation and properties of butyryl
adenylate. Preston T. TarsBert,* F. M.
HUENNEKENS AND BEVERLY W. Gasrio. Depts.
of Biochemistry and Medicine, Univ. of Washing-
ton, Seattle.

Following the definitive work of Berg (J. Am.
Chem. Soc. 77: 3163, 1955), who showed that acetyl
adenylate was an intermediate in the enzymatic
formation of acetyl CoA from acetate, ATP and
CoA, we have investigated the analogous reaction
involving butyrate and the fatty acid activating
enzyme (FAAE). Butyryl adenylate was prepared
in 50-75% yield by the condensation of butyric
acid and adenylic acid at low temperatures with
dicyclohexylearbodiimide as the catalyst. The
compound was purified principally by chro-
matography on cellulose columns with iso-
propanol:water (70:30) as the solvent system.
The material migrated as a single, homogeneous
substance when assayed by paper chromatography
in 3 solvent systems. The purified material was
analyzed for: a) adenine content by 260 mu ab-
sorption; b) total phosphate; c) ester or anhydride
linkages by the HzNOH-FeCl; method; d) free
vicinal hydroxyl groups by periodate titration,
and e) free amino group on the 6-position of the
purine by HNO: deamination. The ratio of these
assays a/b/c/d/e was 1.00/1.00/0.90/0.98/1.07.
When assayed with FAAE and CoA, butyryl
adenylate formed butyryl CoA in the absence of
butyrate and ATP. (Supported by the Office of
the Surgeon General, U. S. Army.)

1205. Distribution of N-acetyl-,-aspartic acid
in brain. Harris H. TALuan (introduced by
Stanrorp Moore). Rockefeller Inst. for Med.
Research, New York City.

The nonprotein, bound aspartic acid that occurs
in large quantities in the brain, but not in other
tissues, plasma or urine of the cat (TALLAN,
Moore AND Stern. J. Biol. Chem. 211: 927, 1954),
has been shown recently to be N-acetyl-L-aspartic
acid (TALLAN, Moore AND Stern. J. Biol. Chem.
In press). Analysis of the brains of animals of
other species has demonstrated the presence of
the compound in high concentrations in mam-
malian and avian brain tissue. The following are
average values, in mg/100 gm of brain, wet weight:
cow 100, cat 103, rat 87, guinea pig 82, duck 110:

oe Hr 2 so ss ee mS 2 ae ee om (ke eee ee om el ee

=
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ume 18

eriza-
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s the
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direct
on the

ityryl
PF. M.
Depts.
ishing-

rT. Am.
acetyl
ymatic
‘P and
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epared
yutyric
s with
|. The
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eneous
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al was
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of the
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)8/1.07.
outyryl
ence of
ffice of

ic acid
iced by
yr Med.

| occurs
n other
‘ALLAN,
’, 1954),
uspartic
'. Chem.
mals of
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1 mam-
ying are
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ick 110:

March 1956

chicken 104. No detectable quantity has been
found in éxperiments with frog, lobster and horse-
shoe crab. In the immature rat, the compound is
present initially in low concentrations (35 mg %
at 8 days), but reaches the adult level by 17 days
of age. A study of the distribution of N-acetyl-
aspartic acid in different parts of cow brain has
shown that it is present throughout, and in the
spinal cord and spinal roots as well. The highest
concentration occurs in the cerebral gray matter.
Approximate values were as follows: medulla 55,
pons 66, cerebellum 86, mesencephalon 88,
thalamus 92, hypothalamus 72, basal ganglia 112,
cerebral gray matter 124 and cerebral white
matter 68.

1206. Muscle purine riboside phosphorylase.
H. L. A. Tarr. Pacific Fisheries Exptl. Station,
Vancouver, Canada.

The riboside phosphorylase previously described
(Federation Proc. 14, 291, 1955) contains 2 major
protein components by ultracentrifuge analysis
and by paper electrophoresis. The phosphorylase
activity appears to be associated with the protein
which migrates most rapidly during electrophore-
sis, but attempts to obtain purer and still active
preparations by starch electrophoresis and by
ion exchange resin chromatography (amberlite
XE 64) have not been successful. Purification is
achieved, as previously described, by rapid pas-
sage through amberlite XE 64 resin conditioned
with 0.2 m phosphate pH 5.5; the enzyme is com-
pletely inactivated if the resin is conditioned
with citrate or acetate buffers. No acceleration of
activity of purified preparations has been observed
by addition of Ca**, Mg** or CSH. Friedkin’s
method (J. Biol. Chem. 207: 257, 1954) has been
used to prepare dicyclohexylammonium de-
oxyribose 1-P in good yield from deoxyguanosine
and dicyclohexylammonium KH POQ,. Dicyclo-
hexylammonium R 1-P has been prepared similarly
but in poorer yield. Ba R 1-P has been prepared
from guanosine in good yield by the general pro-
cedure of Rowen and Kornberg (J. Biol. Chem.
193, 384) except that reaction mixtures are
adjusted to pu 10 before absorbing on Dowex-1
formate columns, washing and eluting at ordinary
temperatures with 0.5 m NaCl. The following
purines are phosphorylated by the enzyme in
presence of R 1-P as judged by liberation of free
orthophosphate: hypoxanthine, xanthine, gua-
nine, 8-azaguanine, adenine and 2,6-diamino-
purine. Purine, 2-thioadenine, isoguanine,
8-chloroxanthine, 8-azaxanthine, 4-amino-5-imid-
azolecarboxamide, cytosine, thymine and uracil
are inactive. The enzyme _ phosphorylates
guanosine in presence of synthetic a-R 1-P but
not in presence of synthetic 6-R 1-P, and therefore
the natural ester has the a configuration. (I am

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

369

indebted to Dr. H. G. Khorana for supplying
synthetic R 1-P esters.)

1207. Citric acid cycle enzymes in normal and
syphilitic rabbit tissues. Henry TavusBer.
Venereal Disease Exptl. Lab., PHS, School of
Public Health, Univ. of North Carolina, Chapel
Hill.

In order to determine the preference of patho-
genic T'reponema pallidum for certain tissues, the
testes, kidneys, liver and heart of normal and
syphilitic rabbits were assayed for aconitase,
fumarase, alpha-ketoglutaric oxidase, pyruvic
oxidase, succinic dehydrogenase and lactic de-
hydrogenase. All enzymes were measured by a
spectrophotometric method, except succinic de-
hydrogenase, which was determined by the
tetrazolium method. Six groups of normal rabbits
and 6 groups of syphilitic rabbits, the latter after
7-66 days of infection before use, were studied.
The average alpha-ketoglutaric oxidase and
aconitase values of syphilitic testes dropped to
3, and the pyruvic oxidase to 4 of the normal
values, both on wet and on dry basis. The enzymes
of the other organs apveared to be unchanged.

1208. Intestinal absorption of glucose in the
rat. W. R. TayLor anp Rosert G. LANGpoNn
(introduced by Lustig HELLERMAN). Dept. of
Physiological Chemistry, Johns Hopkins School
of Medicine, Baltimore, Md.

The mechanism of the absorption of glucose
from the gastrointestinal tract is still obscure.
From the results obtained by in vitro experiments
with rat intestine (WILSON AND WISEMAN. J.
Physiol. 123: 116, 1954), it was concluded that
about half of the absorbed glucose is converted
to lactic acid during the process of absorption.
Since it is questionable that the in vitro experi-
ments reflect fully the physiological process oc-
curring in vivo, the extent of conversion of glucose
to lactic acid during intestinal absorption in the
intact animal has now been examined. Nonisotopic
glucose was administered by intraperitoneal
injection to rats which had been fasted for 12 hr.
After 30 min., 1-C'4-glucose was administered to
one group of animals by stomach tube. Another
group of animals was given the same dose of 1-C'-
glucose by intraperitoneal injection. At varying
times the animals were killed and the liver glyco-
gen isolated and hydrolyzed. The distribution of
the isotopic carbon in the glucose was then deter-
mined. Comparison of the isotope distribution in
the glycogen obtained after stomach feeding with
that obtained after intraperitoneal injection
indicates that cleavage to lactic acid does not play
a quantitatively significant role in the absorption
of glucose by the intestine of the rat in vivo under
the conditions of these experiments.

370

1209. Metabolism of nickel following inhala-
tion of nickel carbonyl. Ratpxu E. TEDEScHI*
AND F. WILLIAM SUNDERMAN. Div. of Metabolic
Research, Jefferson Med. College, Phila-
delphia, Pa.

Nickel balance has been studied in dogs before
and after exposure to nickel carbonyl vapors in
the range of 0.2-1.0 mg/l. for 30 min. Nickel in
the ingesta and egesta was calculated during
several 3-day metabolic periods before and after
exposure. Before the inhalation of nickel carbonyl,
the dogs were found to be in nickel equilibrium,
approximately 90% of the total nickel being ex-
creted in the stool and 10% in the urine. Following
exposure to nickel carbonyl! this relationship was
reversed, approximately 75% of the nickel being
excreted in the urine and 25% in the stool. Al-
though the total amount of nickel in the stool was
slightly increased above the pre-exposure level,
nevertheless the bulk of the inhaled nickel was
excreted in the urine. These studies point to the
usefulness of measurements of urine nickel as a
diagnostic aid in the early detection of exposure to
nickel carbonyl in concentrations too small to
produce acute toxic effects.

1210. Patterns of self-digestion of trypsin and
its derivatives. L. TERMINIELLO,* M. Brer*
AND F. F. Norp. Dept. of Organic Chemistry and
Enzymology, Fordham Univ., New York City.
The self-digestion of trypsin has received at-

tention in recent years in this and other labora-

tories. We were able to show that self-digestion is
considerably inhibited by calcium or manganese

ions (Arch. Biochem. & Biophys. 31: 335, 1951),

or by acylation of the enzyme. This abstract con-

tains the first report of a quantitative study of
peptide bonds split during self-digestion of the
enzyme as a function of the number of trypsin
molecules inactivated in the process. The self-
digestion follows 3 distinct patterns, characteristic
of a) crystalline trypsin, 6) calcium-trypsin or
trypsin stabilized by acetylation and c) trypsin
stabilized by acetylation plus added calcium ions.

In crystalline trypsin 2 distinct phases are recog-

nized: a) rapid inactivation, followed by b) a far-

reaching breakdown of the inactive split-products.

Calcium-trypsin or acetylated trypsin present

remarkably similar breakdown patterns, as in

both there is a linear dependence of the inactiva-
tion of the enzyme on the number of peptide bonds
hydrolyzed. The protective effects of acetylation
and of calcium ions are additive, and acetylated
trypsin, in the presence of calcium, does maintain
its full enzymatic activity even after 2 days of
incubation as 25°C at pu 8.6. A self-digestion
nevertheless occurs, as evidenced by appearance
of a larger number of free amino groups. This sug-
gests the possibility that a partial breakdown of

FEDERATION PROCEEDINGS

Volume 16

the trypsin molecule may occur without impair-
ment of its enzymatic activity.

1211. Metal content of subcellular rat liver
fractions. RatpH E. Tuiers anp Bert L.
VALLEE (introduced by Paunt L. Munson).
Biophysics Research Lab., Dept. of Medicine,
Harvard Med. School, and Peter Bent Brigham
Hosp., Boston, Mass.

While metals are known to exert effects in oxida-
tive phosphorylation, information on the metal
content of subcellular fractions is scanty. The
amount and distribution of manganese, zinc,
calcium, iron, magnesium, sodium and potassium
in rat livers and their subfractions were deter-
mined by spark and flame spectroscopy. The livers
of 25 normal rats were analyzed in groups of 5.
Each liver was perfused with metal-free 0.25 m
sucrose; 5 livers were then pooled, the connective
tissue was removed and fractionation was per-
formed by the differential centrifugation method
of Schneider and Hogeboom. Connective tissue,
nuclei and whole cells, mitochondria, microsomes
and all supernatant fluids were obtained in known
yield. Each fraction was analyzed by a micro-
Kjeldahl method for nitrogen, by flame spectrom-
etry for sodium and potassium, and by spark
spectrography for the other elements. The result-
ing data are presented as amounts and concentra-
tions of the metals in each sample and their
absolute and relative distributions between the
fractions. The differences in metal content of the
various subfractions proved to be statistically
significant. Each showed a definite and char-
acteristic pattern both with regard to the concen-
tration of metals and share of the total amount of
each metal.

1212. Isolation of cellular lipoproteins. Luoyp
E. Tuomas, Nancy C. BruemMMER,* JoHN T,
SmirH* and ARNoLD J. Funckes.* Dept. of
Biochemistry, Univ. of Missouri School of Medi-
cine, Columbia.

Salt-soluble substances were removed from
isolated whole cells of calf liver by repeated homo-
genization with buffered salt solutions and cen-
trifugation at 48,000 xX g. The residue was
resuspended in salt solution and fractionated by
centrifugation at 120, 1100 and 48,000 X g. The 3
fractions had approximately the same nitrogen
and crude lipid content (8.5-9.5% and 40-45%,
respectively), suggesting that essentially one
type of material was present. Detergents,
sequestering agents or alkali do not solubilize this
material, but usually cause a finer dispersion and
chemical decomposition, as do other adverse
conditions, e.g. use of the Waring Blendor. Chemi-
cal decomposition results in a marked difference
among the 3 fractions. Following Bensley and

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ume 16

mpair-

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RT L.
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dicine,
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, zine,
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s of 5.
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. The 3
itrogen
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ly one
ergents,
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Chemi-
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ley and

March 1956

Hoerr (Anat. Rec. 60: 251, 1934), we also extracted
calf liver cells exhaustively with buffered salt
solutions by slow stirring and slow centrifugation,
keeping the structure intact. The residue had
approximately the same nitrogen and crude lipid
content as those from homogenized cells described
above. This residue was, however, composed of
cell ‘ghosts,’ which had a slightly shrunken ap-
pearance and were less dense than unextracted
cells. The nuclei appeared to be almost empty
except for a nucleolus-like mass. This and previous
work suggests that the highly insoluble cellular
lipoproteins are approximately alike in chemical
composition, although very complex. It is of
course not valid to consider these materials as
pure substances nor to attempt to apply molecular
concepts to them.

1213. Effect of carbon dioxide partial pressure
on photosynthetic products in Sedum
leaves. N. E. Totpert anv L. M. Roursauau.*
Biology Div., Oak Ridge Natl. Lab., Oak Ridge,
Tenn.

Sedum leaves, illuminated in air between 0 and
12% C™“O:2, showed marked variation of C' in-
corporation into the free sugars, sucrose and
glycolic acid. At continuously low pressures or
for short experiments at high COz2 partial pres-
sures, sucrose was the major product. Percentage
of C™ incorporated into sucrose at 1% COs or
above dropped with a corresponding rise in free
hexoses. After 3 hr. at 1% CO:, as much glucose
and fructose each were formed as sucrose. Amount
of C' in the corresponding sugar phosphates
showed no major change under any of these con-
ditions. Thus a limiting factor at higher CO: con-
centration was sucrose synthesis, resulting in
hexose accumulation. At CO2 partial pressure of
air or lower, the ratio of either glucose or
fructose to sedoheptulose was about unity. At
higher partial pressure, this ratio increased 100-
fold, mainly because of a decrease in sedo-
heptulose formation. Sedoheptulose accumulation
in Sedum in air may reflect a low partial pressure
of CO: inside these thick leaves. However, sudden
changes in CO: partial pressure also caused ab-
normally high sedoheptulose production. Gly-
colate-C formation did not always parallel
sedoheptulose formation. Great amounts of gly-
colate-C!* were present at low CO: partial
pressures; at 4% COs, there were only traces of
glycolate. This suggests that the glycolate-bi-
carbonate shift (Federation Proc. 14: 292, 1955)
was needed at low CO: pressures, but that at
higher pressures bicarbonate diffusion alone was
sufficiently rapid for photosynthesis.

1214. Papain-catalyzed reactions of glycine-
containing benzoyldipeptides with aniline

D oon sae

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

371

and glycinanilide. Gorpon To.iin,* MILTON

Win1Tz* anv Srpney W. Fox. Chemistry Dept.,

Towa State College, Ames.

Papain-catalyzed reactions of some glycine-
containing benzoyldipeptides with aniline and
glycinanilide have been investigated. When
glycine was adjacent to the benzoyl group and
alanine, valine, leucine or glycine was terminal, a
transamidation reaction was observed. This
resulted in splitting out of the terminal amino acid
residue and the synthesis of benzoylglycinanilide
or benzoylglycylglycinanilide, respectively. When
the glycine residue was C-terminal and alanine,
valine or leucine was interior, there occurred a
direct coupling leading to the synthesis of the
benzoyldipeptide anilide or benzoyltripeptide
anilide, respectively. There was also performed a
series of competition studies in which more than
one carboxoid component was present with glycin-
anilide. In a number of instances it was possible to
interpret the results in terms of the differing rates
with which the carboxoid substrates reacted with
glycinanilide. Examples of these include the
reactions of benzoyl-pt-alanine and benzoyl-pL-
leucine with glycinanilide to give benzoylleucin-
anilide, of benzoylglycine and benzoy]-pt-alanine
to give benzoylglycylglycinanilide, and _ of
benzoylglycine and benzoyl-pt-leucine to give
benzoylleucinanilide. In other reactions, however,
the products did not represent solely the faster
reacting component. For instance, benzoyl-pL-
alanine and benzoyl-pt-leucylglycine yielded a
mixture of benzoylleucinanilide and _ benzoyl-
leucylglycylglycinanilide; benzoylglycine and
benzoyl-pt-leucylglycine gave the same mixture,
although in slightly different proportions; and
benzoylglycine, benzoyl-pu-alanine, and benzoyl-
pu-leucylglycine gave primarily, if not completely,
benzoylleucinanilide. These results should be com-
pared with the fact that benzoyl-pt-leucylglycine
and glycinanilide alone yielded benzoylleucyl-
glycylglycinanilide. A number of mechanisms
capable of accepting for these latter observations
have been considered.

1215. Mechanism of action of phospho-
glucose isomerase. YALE J. Topper. Nail.
Insts. of Health, PHS, Bethesda, Md.

The interconversion of keto- and aldehydo-
sugars and sugar phosphates involves migration
of carbon-bound hydrogen resulting in a net
transfer of such hydrogen to an adjacent carbon
atom. Two mechanistic possibilities present them-
selves: 1) the hydrogen may shift as a hydride
ion in which case the transformation would be
completely intramolecular involving no exchange
with the aqueous solvent; 2) the hydrogen may
migrate as a proton leading to possible exchange
with the solvent. In order to distinguish between

372 FEDERATION PROCEEDINGS

these 2 alternatives in an enzymic reaction, fruc-
tose-6-phosphate (Ba) was treated, in deuterium
oxide, with phosphoglucose isomerase derived
from rabbit muscle. Glucose-6-phosphate (Ba),
the isomerization product which crystallized
from the system, was found to contain 1 atom of
deuterium. The osazone obtained from this ma-
terial was devoid of isotope, indicating that the
deuterium present in the hexose phosphate reside
on carbon-2. These results demonstrate that the
conversion of fructose-6-phosphate into glucose-
6-phosphate, catalyzed by the isomerase, involves
a proton transfer; in this respect the enzymatic
transformation is similar to the alkali-catalyzed
interconversion of glucose and fructose (J. Biol.
Chem. 189: 191, 1951; J. Am. Chem. Soc. 74: 505,
1952). Furthermore, it appears on this basis that
the sequence of events involves an opening of the
hemiacetal ring, followed by activation of the
alpha hydrogen and enediol formation. Whereas
it has been shown that the enzyme has no
mutarotase activity with respect to glucose, it
is not known whether the opening of the hemi-
acetal ring in hexose phosphate is enzyme-
catalyzed.

1216. Reduction of L-xylulose to xylitol by an
enzyme of guinea pig liver mitochondria.
Oscar TousTER, VERNON H. Reyno.tps,* RutH
M. HutcHeson* AND SIEGFRIED HoLLMANN.*
Biochemistry Dept., Vanderbilt Univ. Med.
School, Nashville, Tenn.

Previous reports from this laboratory reported
the isolation of L-xylulose from the urine of normal
humans and guinea pigs (J. Biol. Chem. 215: 677,
1955) and the metabolism of this pentose by
guinea pig liver preparations (Federation Proc.
14: 293, 1955). All of the activity of liver homo-
genates is present in the mitochondria. The
product of the reaction is xylitol, as shown by
isolation of its crystalline penta-acetate. Rupture
of the mitochondria by exposure to hypotonic
solution, followed by centrifugation, showed that
all of the enzymatic activity is in the insoluble
portion. Solubilization of the enzyme is effected
by treatment of the mitochondrial ‘ghosts’ with
butanol. Intact mitochondria reduce 1t-xylulose
in a medium containing phosphate buffer (px 7.5),
magnesium chloride and glutamate. The insoluble
mitochondrial fraction requires, in addition, a
small amount of the soluble portion of the parti-
cles, presumably as a source of glutamic dehydro-
genase. The latter and the glutamate can be
replaced by reduced diphosphopyridine nucleo-
tide, which also serves in the reduction of the
pentose catalyzed by solubilized enzyme. Re-
versibility of the mitochondrial reaction is
suggested by the formation of a small amount of
ketopentose from xylitol; this is enhanced in the

Volume 18

presence of methylene blue. No substrates other
than L-xylulose and xylitol have been found. The
substances tested were: p-xylulose, p-fructose,
p-sorbose, L-sorbose, L-erythrulose, p-sorbitol,
L-arabitol, p-gulitol and p-talitol. The substrate
specificity of the mitochondrial enzyme differ-
entiates it from previously reported enzymes
which catalyze the interconversion of ketoses
and polyols.

1217. Biosynthesis and microestimation of
2,3-diphosphoglycerate (DPG). J. C.
Towne,* V. W. RopweE.i* anv S. GRISOLIA.
Dept. of Medicine, Univ. of Kansas, Kansas City.
The role of DPG as coenzyme for phosphogly-

cerate mutase (PGM) suggests the presence of
DPG in tissues capable of glycolysis. Distribution
studies show DPG only in erythrocytes of most
mammals; those of birds and reptiles contain none.
DPG is biosynthesized from 1,3-diphospho-
glycerate (Rapoport AND LuUEBERING. J. Biol.
Chem. 196; 583, 1952). We have confirmed these
observations by estimating DPG in tissue extracts
using its catalytic effect on PGM. The 1st method
measures rate of appearance and disappearance
of phosphoenolpyruvate at 240 mu, starting with
either purified 3-phosphoglycerate or 2-phospho-
glycerate with PGM and enolase. The 2nd method
measures pyruvate formed from purified 3-phos-
phoglycerate with a bacterial extract and
reactants for citrulline biosynthesis (GrIsoLIa
et al., Biochim. et Biophys. Acta. 17: 277, 1955).
In the standard test, both methods respond
linearly from less than 0.01 um-0.10 um. Crystalline
3-phosphoglycerate salts are contaminated with
DPG. Ion exchange purification permits its use in
these assays. PGM from yeast, streptococcus,
rabbit and chicken skeletal muscle, and from
heart, muscle, kidney, brain, and liver of beef
and sheep is activated by DPG. These results
make it unlikely that PGM from some source can
act without DPG. Chicken erythrocytes contain
no DPG: chicken breast muscle does and also can
biosynthesize DPG either from purified 3-
phosphoglycerate and ADP or from hexosediphos-
phate with aldolase and _ triosephosphate
dehydrogenase. Further work is in progress to
estimate the biosynthetic capabilities of other
tissues for DPG and to investigate other possible
metabolic roles.

1218. Derivatives of 2-amino-1,3,4-thiadi-
azole as niacin antagonists. W. P. Troy,*
A. 8. Stopopa,* S. L. Haturmay* anno J. J.
Oueson. Exptl. Therapeutics Research Section,
Research Div., American Cyanamid Co., Lederle
Labs., Pearl River, N. Y.

The inhibition of certain mouse tumors by some
derivatives of 2-amino-1,3,4-thiadiazole was

“> SDS =" Dh &® =

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March 1956

recently reported from our laboratory. A marked
inhibition of a glioblastoma, a melanoma and a
lymphosarcoma was obtained. Additional tumors
inhibited to varying degrees were a mammary
adenocarcinoma, sarcoma 180 and a leukemia
P1534. The levels of the compounds used ranged
from 25-250 mg/kg/day. Using 2-amino-1,3,4-
thiadiazole and the ethyl and acetylamino deriva-
tives, it has been found that both the tumor
inhibition and toxicity of the compounds could
be reversed by nicotinamide or nicotinic acid.
The inhibition index was found to be about 10 at
dose levels of inhibitors showing a marked car-
cinostatic activity on both the 891 melanoma and
the glioblastoma. The combination of tryptophane
plus pyridoxine did not reverse the inhibitors.
The compounds showed greater activity in rats
and mice fed a synthetic niacin-deficient diet and
under these conditions the effect of rapidly lethal
doses could be reversed by nicotinamide. The
reversals were most efficient when the vitamins
and inhibitor were given simultaneously rather
than several hours apart.

1219. Biological applications of the liquid
scintillation counter. THEODORE T. TRUJILLO
(introduced by W. H. Lanauam). Los Alamos

Scientific Lab., Los Alamos, N. Mex.

Two types of liquid scintillation counters have
made it feasible to detect many radioactive iso-
topes in biological materials with great sensitivity
and ease of sample preparation. The small volume
fast coincidence, internal sample counter is con-
sidered suitable primarily for detecting low energy
beta emitters; however, many high energy beta
and alpha emitters have been detected success-
fully. Several radioactive isotopes in biological
materials were studied and detected with a high
degree of sensitivity when the sample was dis-
solved or suspended directly in the scintillator
solution. The counting efficiencies for the isotopes
investigated were as follows: H*—8%, C'—70%,
Na”—65%, Ca*—55%, S*—55%, and Cs!87—60%.
The alpha emitters U2*?, U25 and Pu?® were de-
tected with 100% efficiency. The removal of
quenching substances present in both fluids
resulted in reliable assays of the low energy betas.
The scintillation solutes used in these investiga-
tions were 2,5-diphenyloxazole (PPO) and 1,4-
di-[2-(5-phenyloxazolyl)]-benzene (POPOP). The
external sample, liquid scintillation counter is
useful for detecting gamma-emitting isotopes.
Whole body counting of small animals has resulted
in turnover studies of the radioactive isotopes
['31, Cg134, Cs!37, Na?? and K®, A special modifica-
tion of the external sample counter, known as the
‘Human Counter,’ may be used for body burden
detection of radium, K® and other gamma-
emitting isotopes in humans and large animals.

D oon ana

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS 373

1220. Myxomyosin, a protein related to struc-
ture and streaming of cytoplasm. PauL
O. P. Ts’o, LutHeR EaGeamMan AND JEROME
Vinocrap (introduced by James Bonner).
Div. of Biology and Div. of Chemistry and Chemical
Engineering, California Inst. of Technology,
Pasadena.

Both gel structure and the rate of protoplasmic
streaming in the plasmodium of the slime mold,
Physarum polycephalum, are visibly affected by
added ATP (J. Gen. Physiol. 39: 325, 1956). A
cytoplasmic protein, myxomyosin, involved in
this response has been extracted from the plas-
modia and studied after being purified by salt
fractionation and differential centrifugation.
Myxomyosin interacts with ATP as shown by
changes in viscosity similar to those demon-
strated by the actomyosin-ATP system. Current
preparations contain 70-80% myxomyosin and
9% PNA. The nucleic acid, while not essential for
the effect of ATP on viscosity, influences the
physical state of myxomyosin in solution. Ac-
cording to viscosity and electron microscopy data
myxomyosin is a rod-shaped molecule with an
axial ratio of ca. 80 to 1 and a length of ca. 4000-
5000 A. A weight-average molecular weight of six
million computed from viscosity and sedimenta-
tion data is in agreement with the molecular
weight calculated from the dimensions and
partial specific volume data. The effect of ATP
on myxomyosin has been investigated by vis-
cosity at variable rates of shear, sedimentation,
flow birefringence, electron microscopy and
electrophoresis. In high dilution myxomyosin does
not change noticeably in shape or size upon ad-
dition of ATP. ATP is bound to myxomyosin as
shown by electrophoresis studies. The viscosity
effects are best understood in terms of an ATP-
myxomyosin interaction which depends upon
concentration.

1221. Effect of diet on uterine weight response
of rats and mice to orally administered
stilbestrol. Ernest J. UMBERGER,* Jack M.
Curtis AND GEeorcE H. Gass.* Div. of Pharma-
cology, Food and Drug Admin., Washington,
DC.

In order to quantitate any differences in the
level of estrogenic activity of tissues from beef
animals fed stilbestrol, known amounts of stil-
bestrol were added to the tissue diets fed im-
mature mice or rats. Although statistically
significant responses over control values could
be obtained when as little as two parts per billion
of stilbestrol was added to beef tissue diets con-
taining about 10% ground laboratory chow, or to
ground laboratory chow, no response in mice and a
greatly diminished response in rats was obtained
with as much as 30 parts per billion added to a diet

374

composed chiefly of horse meat. Studies are in
progress to ascertain the reason for this dimin-
ished response in horse meat diets.

1222. L-threonine, an obligatory precursor of
L-isoleucine in E. Coli. H. E>w1n UMBARGER.
Dept. of Bacteriology and Immunology, Harvard
Med. School, Boston, Mass.

Experiments using mutant strains of micro-
organisms have indicated a close relationship be-
tween the biosynthesis of L-threonine and that of
L-isoleucine. It is well established in Neurospora
crassa and Escherichia coli that exogenous L-
threonine in the medium is converted to L-iso-
leucine. The exact distribution of the 4 L-threonine
carbon atoms within the molecule of L-isoleucine
has been reported. Complete acceptance of L-
threonine as an obligatory intermediate in the
biosynthesis of t-isoleucine has been prevented
by the finding that certain isoleucineless mutants
(class 4) responding to a-ketobutyric acid but not
to L-threonine do, nevertheless, have L-threonine
dehydrase activity (Woop anp GuNsaLus, J.
Biol. Chem. 181: 171, 1949). A re-examination of
several mutant strains of E. coli has now revealed
that the capacity to deaminate t-threonine is
present in class 4 mutants only when ‘deep’ grown
in a peptone medium similar to that recommended
by Wood and Gunsalus. In contrast, L-threonine
dehydrase activity has invariably been found in
extracts prepared from cells grown in minimal
medium for all other strains examined. Thus, in
class 4 mutants, the requirement of L-isoleucine is
due to an inability to form L-threonine dehydrase
in minimal media. It must be concluded that L-
threonine lies directly on the pathway of L-
isoleucine biosynthesis in E. coli. The environ-
mental influence on the formation of L-threonine
dehydrase by the mutant is under study. It is of
interest that L-serine dehydrase activity has not
been affected by the mutation which has occurred
in class 4 mutants. (Supported by Grant 4015
(C2) from the Public Health Service.)

1223. Saturation studies with vitamin B,,
in human. subjects. WALTER G. UNGLAUB,*
O. Neat MILLER AND Grace A. GoLpsmitTH*.
Depts. of Medicine and Biochemistry, Tulane
Univ., New Orleans, La.

Normal subjects, patients with macrocytic
anemia, and a group of patients with miscellaneous
diseases were given vitamin Biz, 50 ug intra-
muscularly, daily for 10 days, followed by 1000
ug daily for an additional 10 days. After a 3-day
interval, a final test dose of 50 wg was admin-
istered. Urinary excretion of the vitamin and
concentrations of free and bound vitamin Bye
in serum were measured at suitable intervals.
There was a significant increase in the concentra-

FEDERATION PROCEEDINGS

Volume 18

tion of free and bound vitamin B,. during the
period of saturation in all subjects, which in most
instances was still present 3 days after the satu-
ration period. In all patients with pernicious
anemia tested thus far, and in some patients with
other types of macrocytic anemia, the maximum
level of bound vitamin B,2. was significantly less
than that attained in normal subjects or in pa-
tients with miscellaneous diseases. During the
period of administration of 50 ug of the vitamin,
urinary excretion increased progressively for the
first 5 days, after which it was maintained at
approximately a constant level in most subjects.
Excretion varied widely among individuals and
among groups at all levels of saturation. No cor-
relation was observed between urinary excretion
of the vitamin and concentration of free, bound or
total vitamin By. in the serum.

1224. Inhibition of yeast alcohol dehydro-
genase by pyridine derivatives. J. vAN Eys
(introduced by W. M. CuiarKk). McCollum-Pratt
Inst., Johns Hopkins Univ., Baltimore, Md.
The reduction of DPN by crystalline yeast

aleohol dehydrogenase is inhibited at relatively
low concentrations by 3- and 4-substituted
pyridine compounds. The inhibition is propor-
tional to the pxa of the ring nitrogen. However,
when the concentration is expressed as the pyri-
dinium ion present, the inhibition follows the
series 3-SO3, > 3-COz2,> 3-CN > 3-CO.Et >
3-CHO > 3-COOH; > 3-CONH: > 3-CH; > 3H
and 4-CH;. The N’-methyl] derivatives follow the
same series, making it likely that the inhibitor is
the pyridinium ion. The relative inhibitory power
of the pyridine compounds toward the reduction
of DPN by ethanol is quantitatively different from
that toward the oxidation of reduced DPN by
acetaldehyde. This difference is most marked in
the case of 4-methyl pyridine which at a con-
centration of 5.10-% m inhibits 50% the reduction
of DPN. The reverse reaction requires 0.1
methy] pyridine for this level of inhibition. This is
different for the pyridine analog of DPN, which
inhibits only the oxidation of DPNH. The py-
ridinium ions will, at low concentrations, activate
the reduction of DPN. This is most marked for
those substances which inhibit also the reverse
reaction. On the basis of these results it is postu-
lated that DPN and DPNH compete, during the
reaction, for the same site. At this site the adeno-
sinediphosphate ribose moiety is bound in
identical fashion for both DPN and DPNH. The
linkage, however, to the protein, of the oxidized
or reduced nicotinamide moiety appears to be
different.

1225. Changes in bile and liver following
prolonged periods of bile duct obstruction.

ay eee a ee a a

ty]
tra
ide

ve 16

the
nost
atu-
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with
mum
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and
cor-
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dro-
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ively
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opor-
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»wing
ction.

March 1956

Harry,M. Vars anp G. CastIGLioni.* Harrison
Dept. of Surgical Research, School of Medicine,
Univ. of Pennsylvania, Philadelphia.

Male Wistar rats were subjected to either 1)
bile duct ligation with subsequent cannulation of
the duct 6-21 days later, or 2) cannulation of the
bile duct, obstruction of the cannula for 6-21
days followed by release of the obstruction. Bile
was collected for periods up to 2 wk. The daily
excretion of fluid, cholates and cholesterol were
determined. With shorter periods of prior ob-
struction the total output of bile constituents
approached normal values in about 1 wk. though
the volume excreted remained high. With the
longer periods abnormal values for volume and
cholesterol persisted during the observation
period. Total cholates showed less change. His-
tologic specimens indicated a rather rapid return
to a nearly normal architecture even after the
longer periods of ductal obstruction. (Supported
in part by contract DA-49-007-MD-143 between
the Dept. of the Army and the Univ. of Penn-
sylvania.)

1226. Cytochromes in denitrifying bacteria.
Leo P. Vernon. Chemistry Dept. Brigham
Young Univ., Provo, Utah.

Fractionation of Micrococcus denitrificans and
Pseudomonas denitrificans extracts reveals the
presence of 2 cytochromes in each bacterium.
Both species contain a cytochrome of the c type
which functions in cyanide-sensitive electron
transport from pyridine nucleotides or succinate
to oxygen, and appear to be the bacterial counter-
part of mammalian cytochrome c. In addition
both bacteria contain a cytochrome which ex-
hibits absorption maxima at 559, 528 and 426 my
in the reduced state and yields pyridine and
cyanide hemochromogens indicating it to be a
cytochrome of the b type, containing proto-
hematin as the prosthetic group. The cytochrome
is oxidized by air, has a low potential, and during
zone electrophoresis at pH 7 on starch, migrates
toward the anode. Exposure of the reduced cyto-
chrome to nitrate in the absence of air, results in
an oxidation of the cytochrome, indicating that
in these 2 bacteria the cytochrome can function in
electron transport to nitrate. An unidentified
nitrate-reducing bacterium has been shown to
contain 3 cytochromes. In addition to a regular
bacterial cytochrome c and the b cytochrome de-
scribed above, it contains a cytochrome which
has absorption maxima at 553, 523 and 419 mu and
forms pyridine and cyanide hemochromogens
which would classify it as a cytochrome of the c
type. This cytochrome can function in electron
transport past succinate, and is assumed to be
identical with cytochrome ¢.

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

375

1227. Inhibition of liver lactic dehydrogenase.
Cart 8. Vestuinc, Hrrosn1 TERAYAMA,*
James R. Fiortni* anp JAMES N. Baptist.*
Div. of Biochemistry, Dept. of Chemistry and
Chemical Engineering, Univ. of Illinois, Urbana.
A number of reagents have been tested to ex-

amine their effects upon the activity of highly

purified rat liver lactic dehydrogenase (LDH).

Most measurements have been made from the

lactate side of the equilibrium. LDH shows no

SH groups amperometrically in aqueous solution,

but about 10 SH groups in 50% ethanol. Iodoace-

tate does not inhibit, but PCMB inhibits rather
slowly and partial recovery can be made with

SH-glutathione. Cysteine and NaS.O, act as

inhibitors and can be reversed partially by weak

oxidizing agents. The most interesting effects have
been obtained with 0.002 m NaS at px 8.5 in
glycine buffer. Under these conditions inhibition
is complete but reversal is accomplished by:

1) dialysis; 2) treatment with Versene, pyro-

phosphate, citrate, o-phenanthroline or other

chelating agents; 3) salting the LDH out of
sulfide solution with ammonium sulfate; 4) treat-

ment with ferricyanide or o-iodosobenzoate; 5)

treatment with ZnCl, (0.002 m). Partial recovery

of activity could be obtained by substituting

Mnt+ or Fet+ ions for Zn*+*, but no recovery was

noted with Nit*+, Cot* or Cutt. These ions, in-

cluding Zn*+, do not affect LDH which has not
been treated with Na.S, but the untreated enzyme
is strongly inhibited by Hg** or Ag*. These effects
seem to indicate the essentiality of certain disul-
fide bonds for activity and present the possibility
that LDH is a metal-enzyme. Further inhibition
studies have confirmed the previous observation
that p-toluene sulfonate acts competitively with
respect to both lactate and DPN*t, but benzene
sulfonate acts competitively with respect to
lactate and noncompetitively with respect to
DPN’.

1228. Anaerobic lipogenesis in fetal liver
slices. C. A. VILLEE, D. D. Hacerman,* R.
KIMMELSTIEL,* J. M. Lorine* anp F. M.
We.uneton.* Dept. of Biological Chemistry,
Harvard Med. School, Boston, Mass.

Liver slices were prepared from 1) fetal rats
obtained by cesarean section near term, 2) rats
18-24-hr. old, 3) rats 1-wk. old and 4) adult rats.
The slices were incubated in buffered saline
containing acetate-2-C!‘, or uniformly C' labeled
glucose in oxygen or nitrogen. After incubation,
the slices were extracted by the method of Folch
and the resulting total lipide extract was analyzed
for radioactivity. Fetal liver slices have a high
rate of lipogenesis aerobically, as evidenced by
the incorporation of carbon 14 into the total
lipide, and an even higher rate under anaerobiosis.

376

In contrast, slices of adult liver or of 18-24-hr. old
liver have a much lower rate of lipogenesis anaero-
bically than aerobically. Fetal liver a day or 2
before birth is also characterized by high glycogen
content, 6-10% of the wet weight. Lipogenesis
from pyruvate or acetate would not yield energy
for survival, but the hydrogenation of the con-
densed acetyl units, by providing acceptors for
the protons released in substrate dehydrogen-
ation, would permit energy release by glycolysis
without concomitant metabolic (i.e. lactic) acid
formation. The well known ability of fetal and
newborn animals to survive prolonged anoxia
may depend upon glycolysis of the great prenatal
glycogen stores with some accumulation of lactic
acid but with even greater conversion of the
products of glycolysis to neutral fat.

1229. Formation of L-arabinose phosphate
by extracts from Propionibacterium pento-
saceum. WresLEey A. VoLkK (introduced by W.
PaRKER ANSLOW, JR.) Dept. of Microbiology,
School of Medicine, Univ. of Virginia, Char-
lottesville.

Cell free enzyme extracts were prepared by
grinding the washed organisms with glass. A
typical experiment contained 1000 um of L-ara-
binose, 500 um ATP, 100 um MgClo, 250 um NaF
and 7.0 ml enzyme extract in NaHCO; buffer
under an atmosphere of CO:. The phosphorylated
pentoses were collected as the water soluble,
alcohol insoluble barium salts. After passage
through a cation exchanger (H* form), they were
chromatographed on Dowex-1 (formate form) and
collected in 10 ml fractions. The following frac-
tions gave a positive orcinol reaction: 1) 42-52,
2) 66-88 and 3) 94-135. Fraction 1 (42-52) was
tentatively identified as adenylic acid on the basis
of its absorption at 260 and 280 mu. Fraction 2
(66-88) was tentatively identified as heptulose
phosphate on the basis of the absorption spectrum
of its orcinol derivative and its reaction in the
cysteine-carbazole test for ketoses. Fraction 3
(94-135) appeared to consist of at least 3 different
pentoses. Tubes 94-108 consisted mainly of an
aldopentose -phosphate which after hydrolysis
could not be separated from p-ribose by paper
chromatography or as a borate complex on Dowex-
1. Tubes 109-128 consisted mostly of a ketopentose
phosphate which after hydrolysis could not be
separated from t-ribulose and tubes 129-135
consisted mostly of an aldopentose phosphate
which after hydrolysis could not be separated
from L-arabinose under the same conditions as
above. The t-arabinose is believed to be phos-
phorylated in the 5-position on the basis of its
stability to acid hydrolysis.

1230. RNA phosphorus turnover in T2r+-
infected Escherichia coli. Exitiot VoLKin.

FEDERATION PROCEEDINGS

Volume 16

Biology Div., Oak Ridge Natl. Lab., Oak Ridge,

Tenn.

Infection of E. coli with bacteriophage T2r+
produces a vigorous synthesis of phage deoxy-
ribonucleic acid and protein but no significant
change in the host’s ribonucleic acid (RNA)
content. There is no general agreement, however,
concerning turnover of RNA after phage infection.
This problem has been reinvestigated under con-
ditions thought to be more definitive than those
previously employed. Thus the RNA contribu-
tion from uninfected bacteria was maintained at
an insignificant level and furthermore, RNA
phosphorus was identified unambiguously with
RNA mononucleotide phosphorus. The results
reveal that medium inorganic phosphate was
incorporated in RNA after phage infection to an
extent some 500- to 1000-fold more than could be
accounted for on the basis of the few remaining
uninfected cells. A marked difference was ob-
served in the kinetics of incorporation in peptone
broth as compared with glucose-synthetic
medium. In peptone broth, a rapid incorporation
of P% into RNA phosphorus quickly ceased,
whereas such incorporation in synthetic medium
continued almost linearly for at least 60 min. In
both cases, it was observed at all sample times
that adenylic and uridylic acid phosphorus had
specific activities some 50% greater than the other
2 mononucleotides, indicating that the incorpora-
tion of isotope was heterogeneous with respect
to the total RNA phosphorus. Partial separation
of the infected cells’ RNA by differential centri-
fugation revealed that supernatant RNA in-
corporated 5-10 times as much P#*Q, as the par-
ticulate fraction RNA, although the latter con-
tained some 60% of the total cell RNA.

1231. 8-hydroxybutyrate and _ acetoacetate
oxidation by heart muscle mitochondria.
R. W. Von Korrr. Dept. of Pediatrics, Heart
Hosp. Research Labs., Univ. of Minnesota,
Minneapolis.

The role of §-hydroxybutyric acid (BOH)
dehydrogenase has been uncertain since the dis-
covery that D(—)BOH and free acetoacetate are
not on the main pathway of fatty acid oxidation.
It has been observed that with washed heart
muscle mitochondria, using malate as sparker at
pH 6.5-6.9, acetoacetate oxidation proceeds very
slowly while acetate, pyruvate, or DL BOH
oxidation occurs readily. Acetoacetate oxidation
ensues when a-ketoglutarate (KG), or malate at
pH 7.4, is added. Several observations suggest that
acetoacetate is oxidized to a considerable extent
via a prior reduction to BOH. 1) Acetoacetate
disappearance frequently exceeds the oxygen con-
sumption if complete oxidation of acetoacetate is
assumed. BOH may be found in the medium. 2)
Anaerobically, acetoacetate and KG yield BOH

[- n,n a oe

noi

is |

me 16

Ridge,

T2r+
eoxy-
ficant
RNA)
vever,
ction.
r con-
those
tribu-
ed at
RNA
with
esults
» was
to an
ild be
aining
is ob-
ptone
thetic
ration
eased,
edium
in. In
times
is had
. other
rpora-
espect
ration
sentri-
A in-
e@ par-
r con-

cetate
ndria.

Heart
nesota,

(BOH)
he dis-
ute are
lation.

heart
ker at
Is very

BOH
dation
late at
st that
extent
acetate
en con-
state is
um. 2)
d BOH

March 1956

and succinate without addition of CoA, DPN or
ADP. 3) D(—)BOH in part is oxidized to com-
pletion and in part to acetoacetate under con-
ditions in which acetoacetate is inert. 4) D(—)
BOH oxidation proceeds readily with little net
acetoacetate formation when KG, L(+)BOH or
succinate is present. 5) L(+)BOH yields little
acetoacetate even in the presence of succinate
plus malonate. Succinyl CoA transferase activity
might be expected to result in free acetoacetate
under these conditions. Succinyl CoA deacylase
and phosphorylating enzyme would oppose aceto-
acetate activation by competing with the trans-
ferase reaction. It is proposed that the classical
BOH dehydrogenase may play an important role
in acetoacetate metabolism as suggested by
observations of Krebs and Johnson nearly 20
yr. ago.

1232. Succinic dehydrogenase system of Bac-
terium tularense. C. L. WapKINs AND R. C.
Mitts (introduced by C. A. Mrutus). Dept. of
Biochemistry, The Univ. of Kansas, Lawrence.
It has been reported previously that the suc-

cinoxidase system of sonic extracts of Bacterium

tularense is a multienzyme system consisting of at
least 3 factors: a heat labile, acid labile, cyanide
sensitive oxidase; a heat labile, acid stable inter-
mediate factor which donates electrons to
methylene blue; and a heat stable, acid labile
dehydrogenase which donates electrons to 2,6-
dichlorophenolindophenol. The following results
on solubilized succinoxidase extracts support the
concept that the succinic dehydrogenase consists
of several components. Heat treated preparations
reduced phenazine methosulfate and 2,6-dichloro-
phenolindophenol but not methylene blue or
oxygen. Indophenol reduction by soluble extracts
was inhibited by 1 X 10-4 m parachloromercuri-
phenylsulfonate and 0.1 m Versene. Mg(II) and

Mn(II) reversed the inhibition by Versene whereas

Ca(II), Fe(II), and Fe(III) failed to do so.

Dialysis overnight against distilled water abol-

ished the oxidase activity. The oxidase activity

was restored 100% by the addition of 10u mM/cc
cysteine and 2 X 107? m Mn(II) and 75-80% by
cysteine and 2 X 10-?m Mg(II). No restoration
occurred with either component alone. The
participation of a flavin in the dehydrogenase is
suggested by the finding that 0.01 m atabrine,

0.005 m riboflavin, and 0.005 m FAD inhibited

indophenol reduction and oxygen uptake while

0.005 m FMN was not inhibitory. When FMN was

added to the atabrine inhibited system, the

oxygen uptake was proportional to the concentra-
tion of FMN. That the reduction of phenazine
methosulfate by the primary dehydrogenase was
not inhibited by 0.01 m atabrine, 0.1 m Versene,
or 1 X 10-4 m parachloromercuriphenylsulfonate
is indicated by the ability of phenazine metho-

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

377

sulfate to reverse the inhibition of oxygen uptake
by these compounds.

1233. Hypothetical structure of cytochrome
oxidase. W. W. Wainio. Bureau of Biological
Research, Rutgers Univ., New Brunswick, N. J.
The hypothesis that cytochrome oxidase might

be a tetrapolymer consisting of 4 identical heme-

protein submolecules is being tested by indirect
means. After preincubation of the enzyme with

NaCN and a small amount of ferrocytochrome c,

it was determined from rate studies that 1 m of

cyanide inhibited at least 2 electron equivalents
of cytochrome oxidase contained in either an in-
soluble particulate preparation or a partially
purified preparation of heart muscle. The test of
cytochrome oxidase activity was its oxidation of
ferrocytochrome c. The number of electron
equivalents of cytochrome oxidase inhibited was
calculated with the aid of the dissociation constant
of the enzyme-inhibitor complex (approximately
1 X 10°). It is thought that a longer preincuba-
tion with more ferrocytochrome c will assure that
the cytochrome oxidase will be in the reduced
state and thus more readily available for combi-
nation with the cyanide. In these circumstances the
calculations are expected to reveal that 1 m of
cyanide will inhibit 4 electron equivalents of
cytochrome oxidase. In manometric experiments

1 molecule of CO displaces 1 molecule of O2.

Since 1 m of O2 corresponds to 4 electron equiv-

alents of cytochrome oxidase, it is expected that

1 mole of CO will inhibit 4 electron equivalents of

the enzyme or the oxidation of 4 m of ferrocyto-

chrome c.

1234. Fatty acid synthesis by a soluble en-
zyme system. Satin J. WAKIL,* Joun W. Por-
TER AND ALISA TiETz.* Enzyme Inst., Univ. of
Wisconsin, Madison.

The synthesis of long chain fatty acids (Cu, to
Cis) from acetate (PoRTER AND GrBson, Abstr. of
papers presented at A.C.S. meeting, Minneapolis,
Sept. 1955) is catalyzed by a combination of at
least 3 different fractions prepared from pigeon
liver. The fractions are obtained by a combi-
nation of ammonium sulfate and alcohol fraction-
ation. The specific activity of the system recon-
stituted with the purified fractions is about 10
times that of the original crude extract. Complete
synthesis of the fatty acids requires, in addition
to the 3 fractions above, the presence of ATP,
Mgtt, Mn*t*, CoA, DPN, TPN, GSH, lipoic acid,
acetate, glucose-l-phosphate, isocitrate and
phosphate-bicarbonate buffer (px 7.0). Glucose-1-
phosphate can be replaced by glucose-6-phos-
phate, fructose-6-phosphate or by a combination
of fructose-1,6-diphosphate, aldolase and glycer-
aldehyde phosphate dehydrogenase. Isocitrate
can be partially replaced by glucose-6-phosphate

378

and glucose-6-phosphate dehydrogenase. The
conversion of acetate to fatty acids is proportional
to both time and protein concentration. Under
optimum conditions up to 50% of the acetate
(1-2um) can be converted to fatty acids. This is
equivalent to a specific activity of 0.05-0.1 um
of acetate converted to fatty acids per milligram
of protein per hour at 38°.

1235. Acid-base properties of rhodopsin and
opsin. GEORGE WaLp AND CHARLES M. Rap-
pinG.* Biological Labs. of Harvard Univ., Cam-
bridge, Mass.

Purified cattle rhodopsin has been titrated to
various pH, irradiated and the px changes fol-
lowed until completed. In this way we have
obtained the titration curves of rhodopsin, of the
immediate (30 sec.) product of irradiation and of
opsin. The exposure of rhodopsin to light between
pH 2 and 8 causes an immediate rise of pH, maximal
at about pu 5. This is the only change of px in the
physiological range. It involves the exposure of 1
new acid-binding group with pk about 6.6, close
therefore to the imidazole group of histidine.
This immediate change is followed in acid or
alkaline solutions by slower changes which
occupy up to 40 min. at 20°C. These are always in
the direction of neutrality. They involve increases
of 5-6 m acid bound at acid pH, and about 7 m base
bound at alkaline po. They are associated with
the irreversible denaturation of opsin, as evi-
denced by loss of the capacity to regenerate
rhodopsin. Whereas rhodopsin is stable for at
least 1 hr. at pH 3.9-9.6 (25°-27°), its product of
bleaching, opsin, is rapidly denatured to both
sides of the narrow range of pH 5.5-7.0. The at-
tachment of opsin to its prosthetic group stabi-
lizes it greatly to acids and bases. (Supported in
part by the Rockefeller Fndn. and the Office of
Naval Research.)

1236. Evidence for an enzyme-amidine inter-
mediate in transamidination reactions.
JAMES B. Waker. Dept. of Biochemistry,
Univ. of Wisconsin, Madison.

An enzyme preparation from hog kidney has
recently been found to catalyze the reversible
transfer of an amidine group from canavanine to
ornithine, with the formation of canaline and
arginine (J. Biol. Chem. 218: 555, 1956). Inasmuch
as Canavanine can serve as a substrate for certain
enzymes whose usual substrate is arginine, e.g.
arginase and argininosuccinase, a reaction mech-
anism was suggested which involves the reversible
formation of the same relatively long-lived
enzyme-amidine complex from either canavanine
or arginine. Additional evidence compatible with
this mechanism of transamidinase action has sub-
sequently been obtained. It has been found that

FEDERATION PROCEEDINGS

Volume ig

the same enzyme preparation catalyzes an arg-
inine-ornithine transamidination reaction. The
radioactive arginine formed by incubating non-
labeled arginine with pt-ornithine-2-C™ and
kidney transamidinase has been identified by
converting it enzymatically to radioactive argi-
ninosuccinic acid. From these data it appears that
the amidine moiety of arginine is in a state of
dynamic metabolic equilibrium in mammalian
kidney and perhaps other tissues. It is proposed
that in kidney tissue arginine, canavanine and
guanidinoacetate can serve as amidine donors via
an enzyme-amidine intermediate, while orni-
thine, canaline and glycine can act as acceptors of
the amidine group from this same intermediate,

1237. Heavy components of human serum,
G. Wa.LLENIvs,* R. Trautman,* E. C. FRANK-
LIN* AND H. G. Kunxkex. The Rockefeller Inst,
for Med. Research, New York City.

About 4% of normal human serum proteins
sediment with so,w = 19S and are referred to as
‘heavy component’. Preparative separation of
lipoprotein free serum by zone electrophoresis
in a starch-supporting medium followed by
analytical ultracentrifugation of each fraction
showed that this heavy component had a peak ‘in
the gamma and also the a2 region. The other main
serum proteins were distributed as follows in
terms of nominal sedimentation rate: ‘88’, a;
‘48’, albumin and a2; ‘5S’, 8; and ‘78’, y, 6 and
a2. The heavy component could be concentrated
relative to the ‘7S’ component by a factor of up
to 23 in each preparative ultracentrifugation,
Electrophoresis and subsequent analytical ultra-
centrifugation of material enriched in heavy
component after 8 ultra-centrifugations yielded
preparations free of albumin with less than 10%
‘7S’ giving again a 2-peaked mobility distribution
of the ‘19S’ component. About 35% was dis-
tributed broadly in the y and £6 regions with
maximum concentration in the y,; region, and 65%
was localized more sharply in a peak in the a:
region. In addition, heavier components with an
Se», w approximately 28S and 448 appeared in the
y2 and y; regions, respectively. A smal] group of
‘12S’ components was widely distributed from the
yi through the a2 region. The y; heavy component
showed by the agar diffusion technique of Ouch-
terlony antigenic differences from the az heavy
component and from the major ‘7S’ component of
serum y-globulin. In pathological sera, the con-
centration of the y; heavy component did not
correlate with elevations in y-globulin, but it was
reduced at least 10-fold in the sera of 2 patients
with agammaglobulinemia.

1238. Glucose metabolism in Streptomyces
griseus. C. H. Wana, J. J. Braty* anp C. M.
Gitmour.* Oregon State College, Corvallis.

lume 16

an arg-
n. The
1g non-
14 and
fied by
re argi-
ars that
state of
umalian
roposed
ine and
10rs via
2 orni-
ptors of
1ediate,

serum,
FRANK-
er Inst,

proteins
d to as
tion of
yhoresis
red by
raction
peak -in
er main
lows in
38’, a1;
, B and
ntrated
r of up
gation.
1 ultra-

heavy
yielded
an 10%
‘ibution
as dis-
1s with
nd 65%
the ay
with an
J] in the
roup of
rom the
ponent
f Ouch-
, heavy
nent of
he con-
lid not
t it was
patients

»my ces
> C. M.
vallis.

March 1966

Time course studies have been carried out on
the utilization of glucose by 12-, 36- and 72-hr.
Streptomyces griseus cells incubated with C14-
labeled substrate in proliferating medium. In
young cells, practically all the administered
glucose was utilized for respiratory and _ bio-
synthetic functions: on the other hand as much as
50% of the substrate glucose was inverted into
fermentation products such as lactic acid in 36-
and 72-hr. cells. The rates of change in specific
activity of the respiratory COs: from cells utilizing
glucose-l1, 2,6 or u-C'4 indicated 1) that the
Embden-Meyerhof scheme and the phospho-
gluconate decarboxylation are the primary break-
down pathways of glucose, and 2) that the Krebs
cyclic processes and CO, fixation of the C; + C;
type follow. From the cumulative radiochemical
recoveries of C!4O,2 obtained in these experiments,
it was possible to calculate the rate of production
of COz from carbon atoms 3 and 4 of glucose in
this organism which in turn revealed that a time
lag of 2-3 hr. is involved in utilizing the ad-
ministered glucose after the latter underwent
transport into the cell. Estimations were made on
the relative significance of the catabolic pathways
of glucose (WanG, Greaa, Forsuscu, CHRISTEN-
SEN AND CHELDELIN. J. Am. Chem. Soc. In press)
for cells of different ages and summarized as
follows: 12- or 36-hr. cells. Embden Meyerhof
pathway 97-98%, direct oxidative pathway 2-3%;
72-hr. cells, Embden Meyerhof pathway 100%.
It also appears that CO, fixation functions more
extensively in the 12-hr. cells which probably
reflects the important role played by the C; + C;
type reaction in active biosynthesis.

1239. Ultraviolet spectra of enzymatically
synthesized polynucleotides. Rospert C.
Warner. Dept. of Biochemistry, New York
Univ. College of Medicine, New York City.
The ultraviolet absorption spectra of the poly-

nucleotides described by Grunberg-Manago,

Ortiz and Ochoa (Science, 122: 907, 1955) have been

measured and the molar extinction coefficients

(referred to polynucleotide phosphorus, « (P))

have been calculated. The e (P) for the adenylic

acid polymer (A) at 258 my and at pH 7, in the
absence of salt, was 61% of that of adenylic acid

(mixture of 2’ and 3’ isomers). This increased to

69% when the ionic strength was raised to 0.2

and further increased to 87% in 6.4 m urea. In

contrast e(P) for the uridylic acid polymer (U)

amounted to 95% of that for uridylic acid and was

almost unchanged by salt concentration or urea.

On mixing the A polymer and the U polymer the

resultant ¢ (P) was 68% of that calculated for the

sum of the separate polymers and 55% of that for
the constitutent mononucleotides. Polymers of
inosinic, cytidylic and guanylic acids also ex-

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

379

hibited lowere (P) values than the respective
mononucleotides, but no pair other than the A
and U polymers showed additional reduction in
e (P) on mixing. The lowe (P) values of these
polymers may thus result from intermolecular as
well as from intramolecular interaction, both
presumably based on hydrogen bonding involving
the purine and pyrimidine bases. By analogy both
may also exist in nucleic acids. Electrophoresis
and sedimentation studies of the A and U poly-
mers and their mixtures support the interpretation
of the intermolecular interaction as leading to a
kinetically stable complex. sible d gastric
1240. Single-carbon transfer reactions and
purine biosynthesis. LEONARD WARREN AND

Jor. G. Fuiaks (introduced by H. C. TrimB1z).

Div. of Biochemistry, Massachusetts Inst. of

Technology, Cambridge.

Two transformylation reactions involving
inosinic acid (IMP) with partially purified chicken
and pigeon liver enzymes account for the in-
corporation of formyl groups into positions 2 and
8 of the purine ring. Enzymatic assays are based
upon the formation or disappearance of 5-amino-
4-imidazolecarboxamide ribotide (AICAR). Either
glycine or glycinamide ribotide (GAR) are 1-
carbon acceptors; with the latter, a-N-formyl-
glycinamide ribotide (FGAR) is formed. Serine
has also been used as a 1-carbon donor. (1) IMP +
glycine + TPNH + H+ = AICAR + serine +
TPNt; (2) IMP + GAR — AICAR + FGAR.
One ethanol and two (NH,).SO, fractions of
chicken liver are required for reaction 1, while
the (NH,)2SO, fractions alone suffice for reaction
2. Both reactions have an absolute requirement
for Kt, a folic acid derivative and a pyridine
dinucleotide. Reaction 1 is TPN specific. t-malate,
fumarate or iso-citrate and Mn*", or a zwischen-
ferment system are also required for the reaction
as written, whereas TPN alone, in substrate
quantities, serves for the reverse reaction. Re-
action 2 requires either DPN or TPN. Anhydroleu-
covorin-A, anhydroleucovorin-B, and isoleuco-
vorin stimulate reaction 1. Isoleucovorin is the
most effective pteridine compound for reaction 2.
Leucovorin is active in purified systems only in
the presence of ATP. Reaction 1 favors IMP
formation but may be studied in both directions.
Reaction 2 favors AICAR and FGAR formation
and attempts at reversal have been unsuccessful.
AICAR has been chemically formylated to 5-
formamido - 4 - imidazolecarboxamide ribotide
(FAICAR). Enzyme preparations from mam-
malian and avian livers catalyze the complete
conversion of FAICAR to IMP.

1241. Effect of sialoadenectomy on growth.
ArtHurR W. WaseE AND Yu SHENG LOUISE

380

Fence (introduced by M. Joun Boyp). Hahne-
mann Med. College, Div. of Biological Chemistry,
Philadelphia, Pa.

Previous observations have indicated the
salivary glands to exert an influence over thyroid
activity, attributed to a decrease in the titre of
TSH as measured by P* uptake. Studies recently
completed indicated that the removal of the
salivary glands from young mice and rats resulted
in reduced rates of growth. No differences were
observed if the carbohydrate portion of the ration
was starch or dextrose. Over a 28-day-test period,
intact control rats showed an average gain in
weight of 3.3 gm/day; sialoadenectomized rats
gained 1.7 gm/day; whereas sialoadectomized rats
receiving 5 ug TSH (Armour) daily, gained 3.1
gm/day. Food efficiency was 37.4, 29.6 and 40.7%,
respectively, in intact controls, sialoadenecto-
mized rats and TSH-treated sialoadenectomized
animals. Carcass analysis performed at the end
of the growth period indicated no significant
differences in the fat and protein composition of
the experimental groups. In vivo studies with S*
L-methionine showed that animals without
salivary glands incorporated 65% as much activity
into liver proteins as did intact animals. Studies
with externally labeled TSH demonstrated that
sialex animals did not degrade the thyrotropic
hormone as readily as did intact animals.

1242. Amine metabolism of Hemophilus para-
influenzae. Ropert H. WEAVER AND Eb-
warp J. Hersst (introduced by E. G. Scumipt).
Dept. of Biochemistry, School of Medicine, Univ.
of Maryland, Baltimore.

Hemophilus parainfluenzae 7901 requires one of
several di- and polyamines for growth. These
amines include putrescine (P), 1,3-propanedi-
amine (PD), spermine (S), spermidine (SD),
and N-(3-aminopropyl)-1,3-propanediamine (Sp-
7) all of which contain a tri- or tetramethylene
diamine constituent. The role of these com-
pounds in the metabolism of this organism is
unknown but the low concentrations necessary for
growth and the results of balance experiments
suggest a vitamin-like function. The purpose of
this investigation was to determine whether the
various growth factors are utilized as such or are
altered by the organism during growth. H. para-
influenzae cells from liquid synthetic medium
containing the various amines were disrupted by
sonic vibration. Protein-free filtrates of the sonic
extracts were steam distilled and the amine con-
stillates were identified by paper electrophoresis.
Extracts of P- or PD-grown cells contain very
small amounts of polyamine but relatively large
amounts of PD. In contrast, cells grown on the
polyamine, Sp-7, contain large quantities of that
amine. These results suggest that P, PD and Sp-7

FEDERATION PROCEEDINGS

Volume 16

are not altered during growth of the organism
whereas S and SD are degraded to PD which
may account for their growth factor activity.

1243. Effects of nucleosides and their deriva-
tives in protein synthesis in cell-free ex-
tracts of pea roots. GEORGE C. WEBSTER,
Dept. of Agricultural Biochemistry, Ohio State
Univ., Columbus, and Div. of Biology, California
Inst. of Technology, Pasadena.

It has been shown (WEBSTER AND JOHNSON,
J. Biol. Chem. 217: 641, 1955) that the incorpo-
ration of radioactive glutamate into the proteins
of cytoplasmic particles from pea roots is mark-
edly promoted by nucleic acid components,
especially by a mixture of the 4 nucleosides
derived from RNA. It has now been found that
the effect is apparently specific for these RNA-
nucleosides, as deoxyribonucleosides or other
non-RNA nucleosides are either inactive or
slightly inhibitory. Although nucleoside-3’-phos-
phates are not as effective in promoting amino
acid incorporation as are free nucleosides, nucleo-
side-5’-phosphates are much more effective. The
greatest promotion of amino acid incorporation
observed thus far is elicited by a mixture of
ATP, GTP, CTP and UTP. A system composed of
these 4 nucleoside triphosphates, a mixture of 17
amino acids and magnesium and potassium ions
results in measurable increases in both the protein
and RNA of the cytoplasmic particles. That
nucleoside triphosphates are acting as precursors
of RNA is indicated by the fact that C!4-adenosine
triphosphate is incorporated into the adenyl
portion of RNA faster than any other precursor
tested, including glucose, formate, glycine, carbon
dioxide, adenine, adenosine, adenylic acid, AMP
and ADP. Inhibitors of protein synthesis (amino
acid antagonists, etc.) are strong inhibitors of
RNA formation. Likewise, inhibitors of nucleic
acid synthesis exert similar inhibitions of pro-
tein synthesis. These findings indicate a close re-

lationship between protein and ribonucleic acid |

synthesis in these cytoplasmic particles.

1244. Enzymatic heterogeneity of crystalline |

trypsin. LeopoLp WEIL AND Marie TELKa.*
Eastern Regional Research Lab. Eastern Utiliza-
tion Research Branch, Agri. Research Service,
U.S. Dept. of Agriculture, Philadelphia, Pa.
An enzyme present in crystalline trypsin prep-
arations capable of hydrolyzing salmine and
clupein and designated as beta-protaminase is
described. In contrast to trypsin, this new enzyme
is not inhibited by diisopropylfluorophosphate or
ovomucoid. Similarly autolysis or pepsin-diges-
tion of crystalline trypsin preparations, which
destroys the tryptic activity, leaves the beta-
protaminase activity unaffected. Heat-inactiva-

ORE cee eo ee eee

fe woe. oeft © ff & &@ 4. eh mm em tlle ele

—

ume 16

‘anism
which
tivity.

eriva-
2€ eX-
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» State
fornia

INSON,
corpo-
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mark-
nents,
osides
d that
RNA-
other
ve or
'-phos-
amino
1ucleo-
e. The
ration
ure of
osed of
e of 17
m ions
protein
_ That
cursors
nosine

adenyl |

cursor
carbon
, AMP
(amino
tors of
nucleic
yf pro-
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ic acid

talline |

‘ELKA.* |
Utiliza- |
Service, |

ia, Pa.
n prep-
1e and
nase is
enzyme
hate or
1-diges-
_ which
e beta-
1activa-

March 1956

tion of crystalline trypsin at acid px is found to
have no effect on the beta-protaminase activity.
This new enzyme is present in crystalline trypsin-
ogen already in its fully active form and the
conversion to trypsin does not result in an increase
of its activity. Crystalline chymotrypsin is free of
this enzyme. Conversion of chymotrypsinogen to
chymotrypsin is not facilitated by the addition
of beta-protaminase. The hydrolysis of salmine
by beta-protaminase is not accompanied by free
arginine formation and results only in peptide
fragments. Autolysis of crystalline trypsin at
pH 8 and subsequent dialysis brings about a 69%
loss of total nitrogen and a total loss of tryptic
activity, but with the complete retention of
beta-protaminase activity. Preliminary specificity
studies of this enzyme are presented.

1245. Lipid-protein complex in egg yolk.
E. O. Wetnman (introduced by Hans Linz-
WEAVER). Western Utilization Research Branch,
Agric. Research Service, U. S. Dept. of Agri-
culture, Albany, Calif.

It has been inferred from isolation studies in-
volving extractions that about half of yolk lipid
is not combined with protein. This hypothesis was
tested by ultracentrifugation studies on unfrac-
tionated yolk. Yolk solutions (5%) were centri-
fuged under conditions in which the chylomicron
lipid aggregates of serum float to the surface and
egg lipoproteins, of which the main component
travels at about 25-30 S; units in density 1.063
(Nicnots et al. Proc. Soc. Exper. Biol. & Med.
85: 352, 1954), remain dispersed in the solution.
The lipid content of the top layer of the centri-
fuged solution was compared with that of a lower
layer. The lipid contents of these layers were
similar, indicating that little if any lipid had
floated to the top. In a sample of ‘sugared’ egg
yolk (glucose removed enzymatically, 15% sucrose
added and homogenized), which had been kept
frozen about 10 months, and in a spray-dried
powder prepared from it, about 5% of the lipids
floated on centrifugation; most of the lipids again
did not rise to the surface. These observations
suggest that nearly all egg-yolk lipids are bound
to protein and that the complexes have densities
greater than that of chylomicrons. Roughly half
of the small amount of ‘floated lipids’ consisted of
triglyceride; phospholipid, cholesterol and traces
of cholesterol ester were also present in this
decreasing order. (The cooperation of Dr. A.
Nichols, Donner Lab., Univ. of California, is
gratefully acknowledged.)

1246. Enzymatic conversion of CDP-choline
and CDP-ethanolamine to phospholipides.
SaMvUEL B. Weiss* anp Eucene P. KENNEDY.
Ben May Lab. for Cancer Research and Dept. of

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

381

Biochemistry, Univ. of Chicago, Chicago, Ill.

The synthesis of CDP-choline (KENNEDY AND
Weiss, J. Am. Chem. Soc. 77: 250, 1955) and of
CDP-ethanolamine is catalyzed by enzymes
(PC-cytidyl and PE-cytidyl transferases) which
are widely distributed in nature. In a 2nd en-
zymatic step, CDP-choline and CDP-ethanol-
amine are converted to lecithin and phosphatidyl
ethanolamine respectively. A 5- to 10-fold stimu-
lation of the conversion of CDP-choline to lecithin
is observed upon the addition of a mixture of
D-a,B-diglycerides to an enzyme system from rat
liver, tested in the presence of surface active
agents such as ‘Tween-20’. This effect is consistent
and quite specific; the addition of the lecithin
from which the D-a,§-diglyceride was derived or
of mixed triglycerides (corn oil) has no effect.
The reaction requires magnesium or manganese
ions; calcium ions are inhibitory. The enzyme is
highly specific for CDP-choline. UDP-choline,
ADP-choline and GDP-choline have been pre-
pared by chemical procedures and found to be
inactive.

1247. 8-Hydroxy-7-methylguanine, a new pu-
rine metabolite in man. BERNARD WEISSMANN*
AND ALEXANDER B. Gutman. Depts. of Medicine,

.Mount Sinai Hosp. and Columbia Univ., New

York City.

Hypoxanthine, xanthine, adenine, 7-methyl-
guanine, guanine, 1l-methylguanine, and N?-
methylguanine have been identified and esti-
mated semiquantitatively in normal human
urine by a recently described method (WEtss-
MANN, BROMBERG AND GutTMAN, Proc. Soc. Exper.
Biol. & Med. 87: 257, 1954; Nature. In press, 1955).
A larger quantity of ‘spot K’, an additional purine
base not identified earlier, has now been isolated
by ion exchange chromatography. Its elementary
analysis, the similarity of its ultraviolet absorp-
tion at pH 0-9 to that of 8-hydroxyguanine, and
its degradation to guanidine and sarcosine estab-
lish that spot K is 8-hydroxy-7-methylguanine, a
compound not previously reported. The daily
urinary excretion is of the order of 1 mg (a min-
imum value), is relatively constant in normal
adults and is not changed significantly on a diet
restricted to glucose or on an antibiotic regimen.
The occurrence of the substance as a normal
human metabolite, thus implied, is confirmed by
the results of feeding glycine-N'* (60% excess,
7 gm) to a human subject (polycythemia vera).
The percentage of excess N!* in urinary 8-hydroxy-
7-methylguanine for a pooled sample representing
the 4 days following dosage is 0.139 (7-methyl-
guanine 0.141, adenine 0.118, hypoxanthine 0.098,
xanthine 0.072, uric acid 0.047). It is of interest
that a 2- to 3-fold increase over normal of the
urinary excretion of this substance occurs in some

382

cases of polycythemia vera, leukemia and acute
gouty arthritis.

1248. Metabolism of some postulated sterol
intermediates. IBpERT C. We.tis. Dept. of
Biochemistry, State Univ. of New York College
of Medicine, Syracuse.

The oxidations of acetate, 6-hydroxy-8-methyl-
glutaric acid (HMG), senecioic acid (SA), A-
hydroxyisovaleric acid (HIV) and cis 6-methyl-
glutaconic acid (MG) have been studied by the
manometric technique in rat liver slices, rat liver
homogenates, CoA-enriched yeast cell (S. ceri-
visiae) suspensions and in homogenates of CoA-
enriched yeast. For the liver slice and liver
homogenate experiments the oxalacetic acid
(OA)-containing medium of Pardee et al. (J.
Biol. Chem. 186: 625, 1950) was employed; no net
oxygen uptake was observed with any of the
substrates in the absence of OA. The vessels
contained 200 mg of tissue as slices or 60 mg of
tissue as homogenate. In the slice experiments
acetate, SA and MG were oxidized; the net oxygen
uptakes (cm. m. 02/10 min.) due to the additions
of these substrates were 4, 3.5 and 2, respectively.
The oxidation of HIV was of questionable sig-
nificance. Only acetate was oxidized by the liver
homogenates, CoA-enriched yeast cells (NOVELLI
AND LipMANN, J. Biol. Chem. 182: 213, 1950) and
yeast homogenates. SA clearly inhibited the en-
dogenous respiration of the whole yeast cells.

1249. In vitro incorporation of C'‘ formate
into the nucleotides of normal and leuke-
mic human leucocytes. WARREN WELLS*
AND Ricuarp J. WinzuLerR. Univ. of Illinois
College of Medicine, Chicago.

Leucocytes from normal individuals and from
patients with acute, chronic granulocytic or
chronic lymphocytic leukemia have been shown to
differ markedly and consistently in their in vitro
incorporation of radioactive formate, and in their
in vitro sensitivity to such metabolic antagonists
as A-methopterin and azaserine. In an effort to
elucidate the biochemical basis for these dif-
ferences, the trichloroacetic acid-soluble fraction
was isolated from leucocytes incubated with C'4
formate and subjected to gradient elution chro-
matography on Dowex-1 formate columns, the
nucleotides and radioactivity in the effluents being
determined. The protein-bound ribonucleotides
were also isolated by Dowex-1 formate column
chromatography after partial purification and
alkaline hydrolysis of the RNA. The specific
activities of the acid-soluble purine nucleotides
were 5-10 times higher than those of the RNA
nucleotides. The formate incorporation into both
the acid-soluble and RNA nucleotides paralleled
that of the total protein fraction, the descending
order of specific activity being acute, granulo-

FEDERATION PROCEEDINGS

Volume 16

cytic, lymphocytic leukemias and normal. No
consistent differences in relative amounts of the
nucleotides in the different types of cells have
been apparent. A-methopterin inhibits by 70-90%
the incorporation of formate into granulocytic
leukemia cells, but has essentially no effect on the
incorporation into lymphocytic leukemia or
normal cells. A-methopterin inhibits formate
uptake by acute leukemia cells by 30-50%. Oc-
casional radioactive components showing no 260
my absorption were noted, but these bore no
consistent relationship to the type of leucocytes
nor to the presence of A-methopterin.

1250. Partial degradation of the benzene ring
of estrone. Harotp WERBIN (introduced by
E. M. K. Geriine). Argonne Cancer Research
Hosp., and Dept. of Biochemistry, Univ. of
Chicago, Chicago, Ill.

The in vivo and in vitro biosynthesis of the
estrogens from carbon-14 acetate has been re-
ported from several laboratories. The mechanism
of this process may be elucidated when methods
are available for the degradation of the labeled
estrogens carbon atom by carbon atom. Pro-
cedures for the partial cleavage of ring D are
known. The partial degradation of the benzene
ring of estrone has now been accomplished per-
mitting isolation of carbon atoms 2 and 4. 2-
Nitroestrone m.p. 183.5-184°C., 4-nitroestrone,
m.p. 270-280°C. (decomposition) and 2,4-dini-
troestrone, m.p. 185-187°C. were synthesized and
subjected to the bromopicrin split. Carbon atoms
2 and 4 were isolated as bromopicrin, tribro-
monitromethane, which was identified by infrared
analysis. After distilling off the bromopicrin from
each degradation and acidifying the reaction
mixture, carbon dioxide was released and pre-
cipitated as barium carbonate. The source of the
carbon dioxide is not certain although carbon
atom 3 appears likely in the degradation of the
mononitroestrones. In the case of 2,4-dinitro-
estrone carbon atom 1 as well as carbon atom 3
probably are oxidized to carbon dioxide. Although
the 3 nitroestrones have been reported in the
literature, the data obtained in this study suggest
that previous workers had isolated mixtures
rather than the pure nitroestrones. Absorption
spectra studies with model compounds indicate
that their assignment of positions to the nitro
groups on the benzene ring may be in error.

1251. Spectrophotometric demonstration of
interaction between proteins and A‘-3-
ketosteroid hormones. ULRIcH WESTPHAL.
Dept. of Biochemistry, Army Med. Research
Lab., Fort Knox, Ky.

The a,f-unsaturated ketonic system at C; which
is essential for the specific biological activity of
most steroid hormones is characterized by light

ouse

8B conse fs: S&S = — DW F&F et = we ms tM

ume 15

aul. No
of the
s have
70-90%
locytic
on the
lia or
ormate
%. Oc-
no 260
ore no
socytes

e ring
ced by
‘esearch
niv. of

of the
xen re-
hanism
ethods
labeled
.. Pro-
D are
yenzene
ed per-
14. 2
strone,
4-dini-
ed and
1 atoms
tribro-
nfrared
in from
eaction
1d pre-
» of the
carbon
of the
linitro-
atom 3
though
in the
suggest
ixtures
orption
ndicate
e nitro

ion of
| A‘-3-
STPHAL.
Pesearch

3 which
vity of
y light

March 1966

absorption of high intensity at approximately
240 mu. An interaction (‘binding’) of this chromo-
phoric group with protein could be expected to
have a definite influence on its electronic con-
figuration, resulting in a possible change of
intensity and spectral position of the ultraviolet
absorption. The absorption maximum of pro-
gesterone, desoxycorticosterone and cortisol in
aqueous solution was found to be at 249, 249, 248
my, respectively. Mixtures of these steroids and
various proteins were prepared in phosphate buf-
fer of pH 7.6. Admixture of human or bovine
serum albumin, 6-lactoglobulin, casein or certain
bovine plasma fractions caused a marked depres-
sion of the molecular extinction coefficients of the
steroids and a slight displacement of the ab-
sorption maximum to shorter wavelengths. These
effects, indicative of interaction, were inversely
related to the ‘polarity’ of the steroids, similarly
as observed in other studies on steroid-protein
interactions (Endocrinology 57: 456, 1955). The
depression of the absorption was far outside the
experimental error which is comparatively large
because of the high UV absorption of the proteins.
No spectrometric indication of an interaction was
given by gelatin, protamine sulfate or egg al-
bumin. The spectrometric procedure permits a
qualitative recognition of steroid-protein inter-
action and an approximate estimation of the
binding strengths. It should be applicable to
other UV-absorbing compounds of suitable
spectral properties. The present results suggest
that the keto group in Ring A is involved in
A‘-3-ketosteroid-protein interaction.

1252. Effect of x-irradiation of mice on thymic
nucleodepolymerase. Patricia P. WEYMOUTH
(introduced by Norton Netson). Clarkson
College of Technology, Potsdam, N. Y.

A single systemic x-irradiation of C57B1 mice
induces changes in nucleic acid depolymerase
activities in distilled water thymic homogenates.
That these changes are not dependent on the
involutional process per se has been demon-
strated by comparison with thymus homogenate
activities after fasting and after hydrocortone
injection. Depolymerase activities were de-
termined by measurement of \ 260 my absorption
by acid-alcohol filtrates after incubation of sub-
strate and homogenate. Desoxyribonuclease
(DNase) was measured at pH 5.5 with Mg** and
10 mg thymus. With RNA, an acid heat labile and
an acid heat stable activity were measured in the
absence of Mg using 0.2 mg thymus. The former
had a pH optimum of 6 (RNase 6) and the latter,
8 (RNase 8). DNA was determined in all homo-
genates and specific activities expressed as dog0/
mg DNA. Fasting of animals after x-ray showed a
similar pattern in all specific activities; a decrease

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

383

at 13 hr. followed by an increase which was ap-
parent at 12 hr. At 24 hr., DNase and RNase 8
were 5 to 6 times normal, while RNase 6 had
doubled. After treatment with hydrocortone to
obtain the same decrease in thymic weight in 24
hr., DNase and RNase 8 were 3 times normal
while the change in RNase 6 was similar to that
in x-rayed mice. With fasting, 48 hr. was neces-
sary to get a comparable thymic weight loss, at
which time only DNase was elevated.

1253. Specific reactions of dinitrofluoroben-
zene with active groups of chymotrypsin.
JoHN R. WHITAKER* AND BERNARD J. JANDORF.
Enzyme Chemistry Branch, Chemical Corps
Med. Labs., Army Chemical Ctr., Md.

In a search of specific inhibitors of ‘active
centers’ in esterases, the reaction of chymo-
trypsin (ChTr) with dinitrofluorobenzene
(DNFB) has been studied under various con-
ditions of pH, temperature and molar ratio of
reactants. The reaction was followed by cor-
relating loss of enzymatic activity with amount
and distribution of protein-bound dinitrophenyl
(DNP) residues. At pu 7.5, 25°C, DNFB: ChTr =
20, 2 m of DNFB per mole of ChTr are bound
without loss of activity; 9 moles DNFB are re-
quired for 50% inactivation. With increasing
pH the number of moles of DNFB required for
50% inactivation decreases, with a minimum at
pH 10.66. At this px, 3.5 X 10-4 m ChTr shows no
appreciable loss of activity in at least 4 hr.;
the addition of DNFB (molar ratio DNFB:
ChTr = 1.2) leads to over 50% inactivation with
binding of less than 1 m of DNFB per mole of
protein. The distribution of DNP amino acids,
isolated under various conditions of reaction,
will be described.

1254. Effect of the glucose blocking com-
pound—2-deoxyglucose—on other fuels in
the extrahepatic tissues. ARNE N. Wick,
BarBARA Britton* anpD RutTH GRABOWSKI.*
Scripps Clinic and Research Fndn., La Jolla,
Calif.

We have previously reported (Proc. Soc. Exper.
Biol. & Med. 89: 579, 1955) that 2-deoxyglucose
rapidly enters the cells of the extrahepatic tissues
(eviscerated-nephrectomized rabbits) in the
absence of insulin and insulin administration
appears to increase the rate of cell entry. The
intracellular transfer of this glucose analog is
associated with a substantial decrease in glucose
transfer and oxidation despite high insulin dosage.
The mechanism involved in the inhibition of
glucose transfer by 2-deoxyglucose does not ap-
pear to be one of simple substitution, but rather a
competition for the same metabolic pathway in
which glucose is blocked by 2-deoxyglucose at

384

some stage in catabolism. Evidence in support of
this is that after the administration of C'!4-labeled
2-deoxyglucose, only trace amounts, if any, are
oxidized. In order to localize the block, the effect
of 2-deoxyglucose on the oxidation of injected
R. A. acetate was examined. It was found that the
glucose analog did not affect acetate oxidation.
These results indicate that the block is associated
with the glycolytic area rather than the oxidative.
2-Deoxyglucose thus appears to offer promise as
a tool for studying insulin action. We have, there-
fore, examined the effect of this glucose analog on
the transfer of galactose and other related hexoses
which respond to insulin action.

1255. Preparation and fate of serum albumin
labeled with C'4 and S**, Dona.p 8. Wiaa@ans,
HERBERT W. RuMSFELD, JR. AND WILLIAM W.
Burr, Jr. (introduced by Hersert C. Tip-
WELL). Dept. of Biochemistry, Univ. of Texas,
Southwestern Med. School, Dallas.

The metabolism of serum albumin has been
studied with a double label tracer technique.
Valine-1-C"4 and methionine-S*® were injected into
male white rats, and after 8 hr., a maximum
amount of blood was collected by cardiac punc-
ture. Serum proteins were separated by ethanol
fractionation followed by starch column electro-
phoresis. The resulting preparation of serum
albumin, which gave a single peak when analyzed
electrophoretically, was injected into a 2nd rat.
After 6 days the rat was exsanguinated by cardiac
puncture and the serum was fractionated as de-
scribed above. Isotope ratios in the 2 albumin
fractions were determined. Intact proteins were
counted in a sequential series of assays, and the
activity attributable to each isotope was cal-
culated following a process of curve fitting. Pre-
vious studies had indicated this method to be
satisfactory for isotope analysis in known mix-
tures without prior separation of the radio-active
amino acids. If individual amino acids in serum
albumin can exchange with similar or different
amino acids in the surrounding medium, it would
be expected that the rate of isotope disappearance
would be unequal. On the other hand, if the
isotopes leave the protein at equal rates, then the
thesis of total synthesis in protein formation
rather than the exchange theory would appear to
be tenable. In this study the net disappearances of
the isotopes from serum albumin were unequal.
(Supported by Contract No. AT-(40-1)-1988 with
the Atomic Energy Commission.)

1256. Reactions of the amino groups of
chymotrypsinogen. Puitip E. WiLcox AND
CuarRLeEs H. CuHERVENKA (introduced by
Wa .TER B. DANDLIKER). Dept. of Biochemistry,
Univ. of Washington, Seattle.

FEDERATION PROCEEDINGS

Volume 16

Conditions have been found at pu 6.9 under
which 1 amino group of chymotrypsinogen reacts
with carbon disulfide to give a mono-dithiocarba-
mate derivative. Kinetic analysis shows that the
remaining 13 amino groups react at a slower rate
by a factor of 200. The recrystallized derivative
has been characterized by chemical analysis, by
ultraviolet absorption and by sedimentation and
electrophoresis. Acidification to pH 3 liberates 1
mole equivalent of carbon disulfide and gives a
protein, in 65% yield after recrystallization,
indistinguishable from native chymotrypsinogen.
When chymotrypsinogen was treated with 0-
methyl-isourea at pH 10.3, a crystalline derivative
was obtained in which the 13.1 lysine residues had
been converted to 12.8 homoarginine residues
with less than 0.2 lysine residues remaining.
Analyses were carried out by ion exchange chro-
matography of acid hydrolysates and by the
Sakaguchi method. The guanidinated protein and
native protein yield equal amounts of DNP-
cysteic acid when treated with DNFB, performic
acid and hydrochloric acid by the Bettelheim pro-
cedure. The guanidinated protein also reacts with
1 m equivalent of carbon disulfide to give a mono-
dithiocarbamate. Acetylation of chymotrypsino-
gen by a me‘uod specific for amino groups gives a
product with 13.9 acetyl groups by acetyl analysis.
Thus the chymotrypsinogen molecule possesses 1
a-amino group which reacts readily with carbon
disulfide but not with 0-methyl-isourea, and 13
e-amino groups which react with 0-methyl-
isourea at pH 10.3 but only very slowly with
carbon disulfide at pH 6.9. All derivatives have
been converted by trypsin into active enzymes.

1257. Aetiology of cardiovascular disease in
choline deficiency. GEorGE F. WILGRAM AND
J. BLUMENSTEIN (introduced by E. E. F. T.
BaEr). Banting and Best Dept. of Med. Research,
Univ. of Toronto, Canada.

Severe choline deficiency may produce fatty and
necrotic changes in cardiac muscle fibers, coronary
medial lipoidosis and aortic sclerosis of the
Moenckeberg type. It has not been established, as
yet, whether those lesions are primarily due to
choline deficiency per se or are secondarily due to
hemorrhagic kidney lesions (HKL) which is
nearly always present in severe choline deficiency.
We have modified and improved the experimental
procedure of renal decapsulation in choline de-
ficiency (Dessau 1947, Hartrorr 1948, BaxTER
1952) to secure rigidly standardized conditions
and thus to determine how the alleviation of
HKL by decapsulation affects the appearance of
cardiovascular disease. Ten choline-deficient rats
have been bilaterally decapsulated. Ten choline-
deficient rats were used as nonoperated controls.
Nine of the 10 bilaterally-decapsulated animals

— > fo ©. Dore = Soe ——— hl oe a ee ee ee ae a a ee

i a a |

—

= 2 nfe <a aw

lume 16

) under
1 reacts
ocarba-
hat the
yer rate
‘ivative
sis, by
ion and
rates 1
gives a
ization,
inogen.
vith 0-
‘ivative
ues had
esidues
\aining.
e chro-
by the
ein and
DNP-
rformic
im pro-
‘ts with
. mono-
ypsino-
gives a
nalysis.
sesses |
carbon
and 13
nethyl-
y with
28 have
zymes.

ase in
AM AND
we ee
esearch,

tty and
yronary
of the
hed, as
due to
due to
1ich is
ciency.
imental
ine de-
3AXTER
ditions
tion of
ance of
nt rats
holine-
ontrols.
animals

March 1956

survived. One died from postoperative ileus with
severe bowel distention. In the nonoperated
group, 6 out of 10 died from HKL. The cardio-
vascular system of the operated animals exhibited
only slight pathological changes. In particular no
aortic changes were discerned 4-month postoper-
atively. In the nonoperated controls, pathological
changes in heart and coronaries were much more
pronounced. Two out of 10 animals exhibited
aortic sclerosis. These results thus confirm and
extend the finding that relief of intrarenal pressure
may tide choline-deficient rats over the critical
period of kidney damage and they show that
under these conditions cardiovascular disease in
choline deficiency may be essentially prevented.
These findings provide strong support for the view
that cardiovascular changes in choline deficiency
are primarily due to severe bilateral haemorrhagic
renal cortical necrosis in the presence of increased
intrarenal pressure.

1258. Enzymatic coagulation of rodent semen.
H. G. WriuramMs-AsHMAN, J. BANKS AND G.
GorTERER (introduced by E. V. JENSEN).
Ben May Lab. for Cancer Research, Univ. of
Chicago, Chicago, Ill.

Vesiculase is an enzyme present in the coagu-
lating gland of rodents which coagulates the pro-
teins of the seminal vesicle secretion. This enzyme
does not coagulate fibrinogen and thrombin will
not clot the seminal vesicle proteins. Vesiculase
has been purified by fractionation with ammonium
sulfate and with acetone and separated from
another, highly active, enzyme in the coagulating
gland which hydrolyzes tosylarginine methyl
ester. The latter substance does not affect vesicu-
lase. The action of vesiculase upon a crude mixture
of the proteins of the seminal vesicle secretion is
most rapid at approximately neutral pH, and is
extremely sensitive to the ionic strength of the
reaction mixture. The enzyme activity is de-
pressed by versene, a,a’-dipyridyl and o-phe-
nanthroline and also by Hg**. The versene inhibi-
tion is counteracted by Mn** and by higher
concentrations of Ca** but not by Mg*t.

1259. Distribution of malic dehydrogenase in
liver cells. J. N. Wittiams, Jr. ano F. T.
pECastro.* Dept. of Biochemistry, Univ. of
Wisconsin, Madison.

Malate is oxidized by isolated mitochrcndria.
Malic dehydrogenase is also reported to be a
soluble enzyme. This dilemma of enzyme location
within cells is also found with other enzymes
(isocitric and betaine aldehyde dehydrogenases).
We have studied the location of malic dehydro-
genase in rat liver cells by the following techni-
ques: Comparison of activity in fractions sepa-
rated by 2 methods (in isotonic sucrose compared

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

385

with 0.88 m sucrose); and studies on serial extrac-
tion of the enzyme from mitochondria with water
(I) and during serial, alternate freeze-thawing and
extraction with water (II). Activity was assayed
by reduction of 2,6-dichlorophenolindophenol at
610 mu. Relative concentrations of enzyme (whole
homogenate = 100) in the fractions were: Isotonic
sucrose method: nucleic plus debris 6, mito-
chondria 11, microsomes 2 and supernatant plus
washings 61; hypertonic sucrose method: 12, 13,
3, 63, respectively. Thus the supernatant con-
tained most of the enzyme, by both methods.
Suspension of mitochondria in water, however,
quadrupled their activity. Freeze-thawing of
sucrose-suspended mitochondria increased activity
2-fold; of water-suspended mitochondria, 1.1-
fold. (I) and (II) gave the following results:
mitochondrial activity of (I) decreased to one-
third after 4 washings; the extracts in (I) gave
no activity after the first washing. (II) gave the
same results as (I). In both cases mitochondrial
activity versus number of extractions leveled
off after the 2nd extraction. Thus it appears that
the enzyme is located in the mitochondria and
that the supernatant activity in the fractionation
experiments probably arises by leaching from the
mitochondria.

1260. Amino acids exposed during autolysis
of pepsin. Martin B. Wiuuiamson Dept. of
Biochemistry, School of Medicine and, Grad,
School, Loyola Univ., Chicago, Ill.
Crystalline pepsin was autolyzed at pu 5.0

(0.1 mM acetate buffer) and at 4° and 37°C. The

reaction chamber was divided into 2 parts by a

cellophane membrane, one compartment con-

taining the enzyme solution, the other containing
an equal volume of buffer. After 48 hr., aliquots of
the dialyzate and the parent pepsin solution were
tested for enzymatic activity by measuring the
liberation of tyrosine from egg albumin solutions.
All the original activity of the parent solution was
retained after autolysis. The dialyzates had no
enzymatic activity by this test. The solutions re-
maining were made alkaline with excess NaHCO;
and an acetone solution of dinitrofluorobenzene
added. After 3 hr. of stirring, the solutions were
made to 6 N HCl and hydrolyzed for 15 hr. The

DNP-products were extracted with ether, dried

and separated from each other on buffered silica

gel columns (0.1 m phosphate buffer, pp = 4.5),

using chloroform as the developing solvent. The

fractions so obtained were identified by paper
chromatography. The products identified included

DNP-leucine (from the N-terminus of pepsin),

DNP-phenylalanine, dinitrophenol and several

DNP-leucine peptides. At 4°C., only dinitro-

phenol was found in the dialyzate, while the

parent solution contained all the products in-

386

dicated above. At 37°C., the principal products
found on both sides of the membrane were DNP-
leucine and DNP-phenylalanine. When these
experiments were repeated at pu 3.5, similar
results were observed, except that DNP-methio-
nine was also found as one of the products.

1261. Role of lactic acid production in glucose
absorption from the intestine. T. Hastincs
Witson (introduced by Cart BacuMan).
Dept. of Biochemistry, Walter Reed Inst. of Re-
Research, Washington, D. C.

It has been shown previously (W1Lson. Biochim.
et Biophys. Acta 11: 448, 1953) that a significant
fraction of the glucose disappearing from the
lumen of the intestine of the rat in vitro is con-
verted into lactate which appears in greater con-
centration on the serosal side. This suggests that
one of the mechanisms of glucose absorption from
the lumen of the intestine is the conversion of
glucose to lactate in the epithelial cells followed
by the discharge of lactate into the portal blood.
The present study deals with the absorption of
radioactive glucose by small sacs of everted in-
testine of the rat and confirms the previous find-
ings. Small amounts of pyruvate and a ninhydrin
reacting material with an Rf corresponding to
that of alanine were found but accounted for less
than 2% of the total radioactivity. Of the initial
radioactivity, 85-90% could be accounted for at
the end of the experiment as either glucose or
lactate. A striking species difference has been
noted in the ability of the small intestine from a
variety of animals to produce lactic acid aero-
bically in the presence of glucose. The jejunum of
the rat and mouse produces large amounts of
lactic acid while that of the hamster, guinea pig
and rabbit produces relatively little. Rat and
hamster jejunum forms lactate from glucose,
fructose and mannose but not from xylose or
ribose. Only small amounts were produced from
galactose.

1262. Alterations in casein by exposure to
ethylene oxide. HERBERT G. WINDMUELLER
AND R. W. ENGEL (introduced by Otar MickEL-
SEN). Dept. of Biochemistry and Nutrition,
Virginia Polytechnic Inst., Blacksburg.

The exposure of vitamin-free casein to gaseous
ethylene oxide for 24 hr. so altered the protein
that when it was fed to weanling albino rats as the
sole nitrogen source they failed to grow. This
growth inhibition was reversed as soon as un-
treated casein was substituted in the diet or when
the treated diet was supplemented with meth-
ionine and histidine. The uptake of ethylene oxide
by casein was followed by the Warburg mano-
metric technique. When individual amino acids
were so tested, only cysteine-HCl revealed a

FEDERATION PROCEEDINGS

Volume 16

rapid uptake, resulting in a compound lethal to
weanling rats when injected subcutaneously,
The Lbs of this unidentified compound was 13
mg of treated cysteine, HC1/50 gm b. wt. Cysteine
(free base) did not undergo this reaction. Elec-
trophoretically, casein exhibited a decrease in
mobility following treatment with the gas. Ad-
dition of the gas to active sites on the protein
was indicated by a decrease in Kjeldahl nitrogen
of 1.4% following prolonged ethylene oxide treat-
ment. The ability of pepsin or trypsin to hy-
drolyze ethylene-oxide treated casein was ap-
parently not impaired.

1263. Micromethods for assay of L-glutamic
acid dehydrogenase and D-amino acid
oxidase in animal tissues. W. J. WINGO AND
WynELLE D. TuHompson.* Dept. of Biochem-
istry, Univ. of Alabama Med. College and School
of Dentistry, Birmingham.

Micromethods, based on measurement of am-
monia by diffusion, according to the Seligsons
(J. Lab. & Clin. Med. 38: 324, 1951) procedure,
have been developed for the above enzymes. The
system for t-glutamic acid dehydrogenase con-
tains (volumes in microliters) 5% tissue homo-
genate, 10-100; 0.3% DPN, 100; ‘coenzyme factor’
(DEWAN AND GREEN. Biochem. J. 32: 626, 1938),
30; 3% methylene blue, 20; M/3 .-glutamate
(adjusted to px 7.3), 50; water, to make 300. The
mixtures are incubated at 37° for 20 min., made
alkaline with potassium carbonate, and rotated
for 1 hr. Ammonia on the collectors is reacted with
5 ml Nessler’s reagent and compared with stand-
ards containing 10 ug of nitrogen, using a Klett-
Summerson photometer with 420 filter. Blanks
containing no tissue are run and appropriate cor-
rections applied. The system for p-amino acid
oxidase contains: 16% tissue homogenate, 10-150;
0.1 m veronal buffer, pH 8.5, 100; 0.3 m pL-alanine
(adjusted to pH 8.5), 50; water, to make 300.
Incubation and determination of ammonia are
done as above. These methods have been tested
on homogenates of liver and kidney from rats and
mice, and of cells of Tetrahymena pyriformis Y.
Responses are linear and sufficiently large that the
methods can be used for assay of relative enzyme
content; however, the protozoa appear to contain
no D-amino acid oxidase. Homogenates of rat and
mouse brain were also assayed for .L-glutami¢c
acid dehydrogenase activity by this procedure.

1264. Paper chromatography of model phos-
pholipide mixtures. R. F. Witter, G. V.
MaRINneETTI,* AND E. Storz. Dept. of Bio-
chemistry, Univ. of Rochester School of Medicine
and Dentistry, Rochester, N. Y.

Solvent systems containing mixtures of diiso-
butyl ketone-acetic acid, methyl isobutyl ketone-

rweaQareosw wa

—_

Tolume 16

lethal to
aneously,
d was 13
Cysteine
on. Elee-
crease in
gas. Ad-
2 protein
nitrogen
de treat-
n to hy-
was ap-

lutamic
10 acid
NGO AND
Biochem-
nd School

t of am-
Seligsons
‘ocedure,
nes. The
ase con-
e homo-
e factor’
6, 1938),
lutamate
300. The
n., made
rotated
ted with
h stand-
a Klett-

Blanks
iate cor-
ino acid
, 10-150;
-alanine
ake 300.
mynia are
n tested
rats and
rmis Y,
that the
enzyme
contain
rat and
rlutamic
ocedure.

l phos-
G.
of Bio-
1 edicine

f diiso-
ketone-

March 1956

acetic acid, or methyl-n-propyl ketone-acetic acid
were found useful for the chromatographic sepa-
ration of phospholipides on unimpregnated filter
paper. Dimethyl, diethyl or methyl ethyl ketones
did not prove satisfactory. Solvents containing
diisobutyl ketone, acetic acid and water were
found to be excellent for the chromatography of
phospholipides on silicic acid impregnated paper.
This latter system permitted the use of larger
amounts of lipides thereby facilitating their de-
tection by spot tests. Model mixtures of lecithin,
lysolecithin, cephalin, sphingomyelin, phospha-
tidic acid and nonphospholipides were com-
pletely resolved in these systems. Acetal phospha-
tide and brain inositol phosphatide prepared by
conventional methods showed very little move-
ment on unimpregnated paper but each was
resolved into at least 2 components on silicic
acid-impregnated paper. These solvent systems
also gave excellent results with circular paper
chromatography.

1265. Inhibition of malic dehydrogenase by
thyroxine. J. Wotrr anp E. C. Wourr (in-
troduced by F. Frreppera). Natl. Inst. of
Arthritis and Metabolic Diseases, Natl. Insts. of
Health, PHS, Bethesda, Md.

It was recently shown by Clarke and Ball
(Federation Proc. 14: 192, 1955) that the apparent
stimulation of succinate oxidation consequent to
the in vitro addition of 10-5 m thyroxine to rat
heart homogenate was an indirect effect resulting
from a decrease in the formation of oxalacetate, a
potent inhibitor of succinic dehydrogenase. In
the present study it is shown that this decrease is
most probably a result of direct inhibition of
malic dehydrogenase by thyroxine. When the
enzyme (60 u—Sigma) is added to 50 um of malate
and 0.1 mg DPN at pu 8.8 (0.1 M tris), L-thyroxine
(3.3-10-5 m) produces at least 60%, and at 10-‘m >
90% inhibition of DPN reduction. Lineweaver
and Burk plots suggest that the inhibition is
competitive in type with respect to both malate
and DPN. Thyroxine produces no significant
shift in the UV absorption spectra of DPN or
DPNH in the concentrations employed. L-tri-
iodothyronine appears to be as active as thyroxine,
whereas triiodothyroacetic acid, p-thyroxine,
D,L-tetrachlorthyronine, p,L-3,5-diiodo-3’ ,5’-di-
methylthyronine, and butyl 4-hydroxy-3, 5-diiodo-
benzoate show less effect (about 50% inhibition
at 10-4 m). pi-thyronine, 3,5-diiodothyronine,
L-diiodotyrosine, pL-tetranitrothyronine, DL 3,5-
diiodo-3’,5’-dinitrothyronine and 2,4-dinitro-
phenol show insignificant or no inhibition at 10-4
M concentration.

1266. Synthesis of lactose: unequal distribu-
tion of C'4 in glucose and galactose. H. G.

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

387

Woop anp P. ScuamsyE. Dept. of Biochemistry,

Western Reserve Univ. School of Medicine, Cleve-

land, Ohio, and Dept. of Spec. Pathology, Royal

Veterinary and Agricultural College, Copenhagen,

Denmark.

Other investigators found nearly equal amounts
and a similar distribution of C'4 in the hexose
moieties of lactose after administration of glu-
cose-C!4, However Schambye et al. (Federation
Proc. 12: 264, 1953) observed in rabbits given
labeled acetate, pyruvate, butyrate or glucose a
non-identical C! pattern of the hexoses. In the
present studies CH;C'4OOH has been used, the
glucose and galactose separated on a cellulose
column and subsequently degraded with Leuco-
nostoc mesenteroides. In lactose from perfusion
of cows udder 95% of the C'4 was found in the
galactose and 5% in the glucose. Further, the
distribution in the galactose was unsymmetrical:
C-4 having approx. 4 times more C4 than C-3,
with small amounts in C-1 and C-2, and very little
in C-5 and C-6. In in vivo experiments after a
single intravenous injection the glucose and
galactose had approx. the same total activities,
but the C! distribution in the galactose was less
symmetrical than in the glucose, C-4 > C-3 >
C-2 > C-1, C-5 and C-6 having little activity.
The same striking difference between glucose and
galactose activities as in the perfusion experi-
ments was obtained by injecting acetate for $
hour into the main arterial supply of a cows udder
and collecting the milk immediately. Our results
thus raise 2 major problems /) the unsymmetrical
labeling of the galactose and 2) the high activity
of the galactose as compared to that of the glu-
cose. Since the Krebs cycle and the glycolytic
scheme alone cannot account for the marked
asymmetry of the galactose other pathways must
be involved. The lower activity of the glucose
moiety may be explained if it derives from free
glucose (in equilibrium with blood glucose), while
the galactose derives from phosphate esters.

1267. Differential metabolic and necrotizing
responses of tumors to podophyllin and
colchicine derivatives. Mark Woops AND
Dean Burk. Natl. Cancer Inst., Natl. Insts. of
Health, PHS, Bethesda, Md.
Acetylpodophyllotoxin-w-pyridinium chloride

inhibits in vitro anaerobic glycolysis (Q%%co.) of

$91 mouse melanoma slices in Krebs-Ringer
bicarbonate by potentiating the anti-insulin
hormones that inhibit the hexokimase system

(J. Nat. Cancer Inst. 16: 351, 1955). Continued

studies show that S91 melanoma glycolysis is also

inhibited, over the same mechanism, by podophyl-

lotoxin, alpha-peltatin, and beta-peltatin (70-

90% by 200-400 ppm). Quercetin is less active

(50% inhibition at 440 ppm), and iso-podophyllic

388

acid and podophyllotoxin glucoside are inactive.
The in vitro activities of these compounds roughly
parallel their tumor-necrotizing properties as
found by Leiter, Waravdekar et al. The in vitro
glycolysis of the relatively insulin-insensitive K-2
carcinoma is virtually uninhibited by all of the
foregoing materials except quercetin at high
concentration (30% inhibition at 440 ppm).
However, certain alcohol-soluble materials ob-
tained by fractionation of commercial podophyllin
or of fresh tissues of Podophyllum peltatum strongly
inhibit Q’%co, of both S91 and K-2 tumors (90%
at 250 ppm). At low concentrations (3-40 ppm)
some of these fractions have a selective anti-
insulin activity in melanoma that is 3-10 times
greater than the activities of the pure inhibitory
compounds, various mixtures of which did not
show synergistic activity. Two tumor-necrotizing
colchicines, colchicine and trimethylcolchicinic
acid methyl ether-d-tartrate, and 2 isomers
thereof, inactive with respect to necrotization,
and a glucoside of desmethylcolchicine, produced,
at 200-400 ppm, little inhibition (-15%) of
Q*®co, in either 891 or K-2 tumors. The data
show that the podophyllins and colchicines may
act differently on tumor cell metabolism, in spite
of any similar or dissimilar in vivo necrotizing
effects that may be involved.

1268. Enzymatic synthesis of complex
pteridine coenzymes. BARBARA E. WRIGHT
(introduced by W. W. Kreuuey). Natl. Insts. of
Health, PHS, Bethesda, Md.

Previous work has demonstrated a requirement
for catalytic levels of polyglutamyl pteridines in
the formation of glycine from serine in the pres-
ence of cell-free extracts of Clostridium H.F.
(J. Am. Chem. Soc. 77: 3930, 1955). With some of
these fluorescent pteridines (‘Co C’) amino acids
other than glutamic are released by acid hy-
drolysis. It has recently been found that similar
fluorescent compounds are obtained enzymatically
when a teropterin preparation (gift of H. Broquist,
Lederle Labs.) is incubated with cell-free extracts
of C. H.F. in the presénce of serine and ortho-
phosphate. The formation of the fluorescent
compounds does not depend upon Mnt*, Bs and
DPN, which are required in addition to ortho-
phosphate for the further formation of glycine
from serine. The complex pteridines formed were
subjected to acid hydrolysis and the amino acids
determined quantitatively by formation of the
DNP derivatives (A. L. Levy, Nature 174: 129,
1954).

1269. Trehalose in insects. G. R. Wyatt anp
G. F. Kaur (introduced by J. S. Fruton).
Dept. of Biochemistry, Yale Univ. School of
Medicine, New Haven, Conn.

FEDERATION PROCEEDINGS

Volume 1§

The blood of silkworms contains a nonreducing
carbohydrate which yields only glucose on hydrol-
ysis (ef. Kuwana, Jap. J. Zool. 7: 273, 1937),
Using pupae of the silk moth Telea polyphemus,
this carbohydrate has now been isolated in crystal-
line form and identified as trehalose (dihydrate)
by the following characteristics: m.p. 97°C,
la]lS = +182 (H20; c, 1), agreement with authentic

trehalose on paper chromatography, formation of

an acetate agreeing in m.p. (78-79°C) and crystal
form with trehalose octaacetate. Trehalose is the
major carbohydrate in blood of both larvae
and pupae, occurring at approximately 5 mg/ml,
The level of glucose is less than 75 as great; in
pupal blood, some glycogen is also present.
Components corresponding chromatographically
to trehalose have also been found in all other
insect species yet examined, which include repre-
sentatives of 5 orders (Platysamia cecropia,
Bombyx mori, Diprion hercyniae, Aedes aegypti,
Drosophila repleta, Oncopeltus fasciatus, Tribolium
confusum). Apart from its occurrence in certain
mannas which are insect exudates, trehalose hag
not hitherto been reported in insects; it now ap-
pears, however, that its presence may be wide-
spread in this group. In view of this finding,
determinations of glycogen and reducing sugar
are not sufficient, as has often been assumed, to
estimate the carbohydrate reserves of an insect.
Preliminary experiments indicate that the metab-
olism of trehalose in silk moth pupae may involve
a phosphorylase (cf. Freresacqun, C. R. Acad.
Sci. 213: 88, 1941). (Supported by a grant from the
Public Health Service.)

1270. Microassay for fibrinogen used for serial
determinations following stress. H. D.
Wycorr, JuLIA AGEE AND JANIS PARSONS (in-
troduced by W. Know.tton Hatz). Dept. of
Oncology, Med. College of Georgia, Augusta.

An assay for plasma fibrinogen was developed
which requires only 0.05 to 0.1 ml of citrated
plasma for a determination. The plasma is diluted
with a solution of thrombin in alcoholic saline.
The resulting clot is washed, dried and weighed
on a microchemical balance. Blood samples were
taken by heart puncture under ether anesthesia,
Two-tenths ml blood samples can be taken from
the same rats every 4 hr. with no change in the
fibrinogen level. Autolyzed Serratia marcescens
cultures were injected subcutaneously into various
groups of rats and the plasma fibrinogen level
was measured every few hours following the in-
jection. The extent of the rise in fibrinogen under
these conditions increases slowly with increase in
dose of toxin. With a given dose the maximum
level reached is independent of the starting level
in the respective rats. Over a wide dose range the

ine

Oxi

ume 16

ducing
1y drol-

1937),
hemus,
rystal-
rdrate)

97°C,
shentic

tion of

crystal
is the
larvae
ng/ml,
eat; in
resent,
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other
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egypti,
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sertain
se has
OW ap-

wide-
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om the

serial
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arious
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ase in
imum
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March 1956

fibrinogen starts to rise about 4 hr. after injecting
the toxin and reaches a maximum in about 15
hr. At low levels of toxin a 2-stage rise is observed:
the fibrinogen rises slightly above normal in 5 hr.,
remains constant for the next 3-4 hr., then rises
sharply during the next 5 hr.

1271. Substrates and inhibitors for xanthine
oxidase. JAMES B. WYNGAARDEN (introduced
by V. E. Price). Natl. Inst. of Arthritis and
Metabolic Diseases, Natl. Insts. of Health, PHS,
Bethesda, Md.
2,6-Diaminopurine has been found to undergo

oxidation slowly in the 8 position in the presence
of large amounts of xanthine oxidase and may now
be included among the purine substrates of this
enzyme. 0.2 um of certain purines were incubated
in air at 25° in 3 ml of M/10 TRIS buffer px 8.26
with 12 mg of purified milk xanthine oxidase in
the presence of catalase. The following initial
rates of oxidation were observed: hypoxanthine
8.9 um/hr., xanthine 11 um/hr., adenine 0.068 um/
hr., isoguanine 0.067 um/hr., 2,6-diaminopurine
0.017 um/hr. Hypoxanthine and xanthine oxida-
tions to uric acid were followed at \ = 292 myz
(40.D. = +0.089/y/ml and +0.066/y/ml, re-
spectively), adenine and isoguanine oxidations to
2,8-dihydroxy-6-aminopurine at A’ = 305 my
(60.D. = +0.114/y7/ml and +0.087/7/ml, re-
spectively), and 2,6-diaminopurine oxidation to
2,6-diamino-8-hydroxypurine at A = 292 my
(AO0.D. = +0.026/7/ml). Generation of respective
8-hydroxy, or 2,8-dihydroxy, derivatives was sug-
gested by the observation of theoretical O.D.
changes at wavelengths mentioned above, and
was confirmed by demonstration of spectra of the
appropriate derivatives at pH 2, 6 and 9. Guanine
incubation slowly yielded uric acid as product,
indicating presence of guanase activity in milk
xanthine oxidase. Inhibition of hypoxanthine
oxidation by adenine, guanine, isoguanine or
2,6-diaminopurine was demonstrated. Under
conditions where hypoxanthine oxidation gave an
average initial rate change of +AO.Dam =
0.069/min., the presence of the 2nd purine in
6:1 m ratio (hypoxanthine 37/ml) yielded hypo-
xanthine oxidation rates as follows: + adenine =
0.006/min., + guanine = 0.034/min., + isogua-
nine = 0.007/min., + 2,6-diaminopurine =
0.029/min. Studies are underway to determine the
nature of the inhibitions.

1272. Calcium requirement of Azotobacter.
OrviLLE Wyss anp Mina-Yu Wane Cuv.
Dept. of Bacteriology, Univ. of Texas, Austin.
The introduction of the chelating agent, eth-

ylenediamine tetra-acetic acid (EDTA) into the

culture medium for Azotobacter agile resulted in

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

389

growth inhibition which could be relieved by the
incorporation into the medium of additional
quantities of both calcium and magnesium salts.
Strontium at equimolar levels substitutes for
calcium in relieving the inhibition of growth by
excess EDTA as does pyridoxamine (1 ug will
replace 50 ug of calcium) and mixtures of amino
acids (3 wg each of phenylalanine and arginine
replace 50 ug of calcium) but such additions do not
modify the magnesium requirement. The addition
of high concentrations of either strontium or
magnesium salts in the absence of EDTA will
also result in growth inhibitions of azotobacter
that can be relieved by the addition of calcium
at a molar ratio of about 1 to 100 for strontium and
1 to 10,000 for magnesium. The competitive ratio
can be altered in that the amount of calcium
required to reverse the inhibition can be reduced
by supplying intermediates of the tricarboxylic
acid cycle and in some cases additional calcium
can be dispensed with entirely if small amounts
of amino acids are added. The relative effects of
these additions are difficult to evaluate since the
addition of many of the organic substances
stimulates the growth rate of azotobacter in the
absence of interference with calcium nutrition.
When these difficulties are resolved experiments
of this type should yield information on the direct
and indirect functions of calcium in the nutrition
of these microorganisms.

1273. Effect of added citrate upon solubility of
tricalcium phosphate in urine. CLaupE L.
YARBRO* AND JAMES C. ANDREWS. Dept. Bio-
chemistry and Nutrition, School of Medicine,
Univ. of North Carolina, Chapel Hill.

The ability of citrate to complex calcium has
considerable implication in the treatment and
prevention of urinary calculi. This is especially
true since several investigators have demonstrated
a lowered citrate excretion in chronic stone
formers. For these reasons the effect of added
citrate upon the solubility of tricalcium phosphate
in urine was investigated. Citrate in the amount of
1, 2 or 5 gm/l. was added to urine and the urine
equilibrated over a 6-hr. period with solid tri-
calcium phosphate, 2 gm/100 ml of urine. At regu-
lar intervals of time, aliquots were taken for the
determination of phosphate. The equilibration
curves so produced were compared with those
obtained with urine to which no citrate had been
added. In general the effect of the added citrate
was to abolish or delay the initial drop in phos-
phate concentration which was previously noted
(J. Urol. 72: 930, 1955). The phosphate solubility
was increased by 0.6-1.0 mm/I. at the level of 1
gm/l. of added citrate and by 1.0-4.8 m/l. at
the 5 gm/1. level of added citrate. These increases

390

in phosphate solubility, ranging from 4 to 20%
over those obtained on urines where no citrate was
added, are equivalent to }-{ of the molar amount
of added citrate. (Supported by a grant (A-248)
from the Natl. Inst. of Arthritis and Metabolic
Diseases, PHS.)

1274. Uridine diphosphate conjugate as
growth requirement for Paramecium
aurelia, variety 4, stock 51.7 (sensitives).
Grorce R. Youna* anp W. J. vAN WAGTEN-
pONK. Dept. of Chemistry and Dept. of Zoology,
Indiana Univ., Bloomington.

P. aurelia, var. 4, stock 51.7 sensitive grow and
multiply in an axenic medium consisting of 17
amino acids, 8 water soluble vitamins, 11 salts,
stigmasterol and 2 fractions from a water extract
of Fleischmann’s ‘activated dried yeast.’ These
fractions are the precipitate and supernatant of a
50% acetone fractionation of the water extract of
yeast. Both yeast fractions are necessary for
growth of this stock. The acetone supernatant
has been further purified by a perchloric acid
(5%) precipitation, a barium ethanol (76%)
precipitation, an acid ethanol (85%) precipitation
and an adsorption on charcoal. The active fraction
eluted from the charcoal by ammonia-ethanol-
water has been identified as a uridine diphosphate
conjugate containing an unidentified amino
sugar, Uridine diphosphate glucose and uridine
diphosphate acetylglucosamine have been tested
for growth-promoting activity and have been
found to be slightly stimulatory. Quantitative
tests on the active fraction give the following
molar ratio of uridine to phosphate to hexosamine:
1:1.8:0.7. The total phosphate to labile phosphate
molar ratio is 2:1. (Supported by a contract be-
tween Indiana Univ. and the Office of Naval
Research (Contract number 60-NR 18010) and by
grants from the Natl. Insts. of Health (No.
C-2160(c)), Natl. Science Fndn., the Rockefeller
Fndn., and Indiana Univ.)

1275. Labile-reduced derivatives of pteroyl-
glutamic acid. Siamunp F. ZAKRZEWSKI* AND
CuarLEs A. Nicuou. Dept. of Pharmacology,
Yale Univ., New Haven, Conn.

Reduced derivatives of pteroylglutamic acid
(PGA) and 10-formyl-PGA were synthesized for
comparison with labile compounds derived
enzymatically from PGA or p-aminobenzoic acid
in bacterial systems. Similar labile precursors of
citrovorum factor occurred in pigeon liver, mouse
leukemic cells and urine of patients receiving
PGA. Available samples of dihydro-PGA and
tetrahydro-PGA had low Streptococcus faecalis
activity and contained up to 50% of p-amino-
benzoylglutamate (identified chromatographi-
cally). Samples of tetrahydro-PGA, prepared

FEDERATION PROCEEDINGS

Volume 1§

according to O’Dell et al. (J. Am. Chem. Soc. 69:
250, 1947), contained only 1% folic acid activity
(S. faecalis) when assayed immediately after
hydrogenation (4 atoms hydrogen per mole of
PGA). Upon aging or catalytic oxidation, the
microbial activity for both S. faecalis and Leuco-
nostoc citrovorum and the content of the dia-
zotizable amine increased; at least 5 fluorescing
compounds occurred on paper chromatograms
and no PGA could be detected on bioautograms,
One compound in the oxidized samples, active for
Im. citrovorum, had the same R; value (0.38,
using 0.1 m KH.PO,) as an unidentified compound
derived enzymatically from PGA. Synthetic
tetrahydro-PGA was determined quantitatively
by titration with iodine (4 equiv. of iodine per
mole of tetrahydro-PGA were reduced by solu-
tions handled under paraffin oil). By anaerobic
paper chromatography (freshly autoclaved buffer
ascending through a column of toluene) 2 zones,
possibly diastereoisomers of tetrahydro-PGA,
capable of reducing iodine, were obtained. The
extreme lability of tetrahydro-PGA necessitates
reservations in assigning metabolic activity to
impure preparations presumed to be tetrahydro-
PGA.

1276. Thymine-5-bromouracil ‘exchange’ in
deoxyribonucleic acid of Escherichia coli.
STEPHEN ZAMENHOF, KENNETH RiIcH* AND
RosatiE DE Giovanni.* Dept. of Biochemistry,
Columbia Univ., New York City.

The cells of Escherichia coli strain I can continue
to replace the thymine in their DNA by 5-bromo-
uracil (up to 48%) even after they cease dividing
(J. Biol. Chem. In press). Under proper con-
ditions, the cells which have started to replace
thymine by 5-bromouracil will continue to do so
even when resuspended in saline containing an
excess of thymine but no 5-bromouracil; evidently,
the supply of the latter (as such or in another
form) is inside the cell. The mechanism may
involve formation of precursors and/or adaptive
enzymes which favor introduction of 5-bromo-
uracil. The size of the ‘exchanging’:fragment is at
present unknown. Incubation in broth containing
5-bromouracil beyond 24 hr. does not change
significantly the 5-bromouracil content in DNA;
whereas the addition of fresh such broth (which
allows cells to divide) decreases this content,
possibly by dilution of precursor due to cell
division. When cells containing 5-bromouracil in
their DNA are grown in broth free of 5-bromo-
uracil, the rate of disappearance of the latter
from the DNA is 2.5 times faster than the rate of
de novo DNA synthesis; this indicates that the
converse phenomenon (replacement of 5-bromo-
uracil by thymine) now takes place. The viscosity,

st:

lume 1§

soc. 69;
ctivity

after
nole of
ym, the
Leuco-
1e dia-
rescing
ograms
grams,
tive for

(0.38,
npound
nthetic
atively
ine per
y solu-
aerobic
| buffer
zones,

sitates
yity to
thy dro-

ge’ in
a coli.
* AND
mistry,

mtinue
bromo-
ividing
r con-
replace
» do so
ing an
dently,
nother
n may
laptive
bromo-
nt is at
taining
change
DNA;
(which
yntent,
to cell
acil in
bromo-
latter
rate of
at the
bromo-
cosity,

March 1956

the heat stability and the U.V. absorption spec-
trum of DNA containing 5-bromouracil have been
investigated: they could not be distinguished
from the ‘normal.’

1277. Tyrosine metabolism in liver: a require-
ment for catalase in oxidation of p-hydroxy -
phenylpyruvie acid. Vincent G. ZANNONI
AND Bert N. La Du (introduced by BERNARD
B. Bropte. Lab. of Chemical Pharmacology,
Natl. Heart Inst., Natl. Insts. of Health, PHS,
Bethesda, Md.

The enzyme system of dog liver which catalyzes
the oxidation of p-hydroxyphenylpyruvic acid
to homogentisic acid has been resolved into 2
protein components, called fraction A and B
(J. Biol. Chem. In press). The components were
separated by treatment with chloroform, frac-
tionation with ammonium sulfate and adsorption
on calcium phosphate gel. Fraction A has been
identified as catalase since the activity of frac-
tion A is proportional to its catalase content and
can be replaced by purified beef or rat liver
catalase. The substrate is not oxidized to homo-
gentisic acid by either catalase or fraction B alone.
Catalase in the presence of enzymatically gen-
erated peroxide does not catalyze this oxidation.
However, in the presence of fraction B a few
gammas of catalase is sufficient to oxidize 5 um
of p-hydroxyphenylpyruvic acid in 1 hr. The
requirement for catalase appears to be specific;
hemoglobin, ferritin and cytochrome c cannot
replace it. The role of catalase in this oxidation
will be discussed. Upon purification of the enzyme
system ascorbic acid no longer stimulates the
oxidation of p-hydroxyphenylpyruvic acid. How-
ever, reduced 2,6-dichlorophenolindophenol is
still effective. This observation suggests that
ascorbic acid may act indirectly through an agent
inactivated or removed during purification.

1278. Manometric determination and speci-
ficity of trypsin and related proteases.
E. Apert ZELLER, ANIMA DeEvi* AND JoHN A.
CarBon.* Dept. of Biochemistry, Northwestern
Univ. Med. School, Chicago, Til.
Benzoyl-t-arginine ethyl ester (BAEE) is a

substrate of trypsin (Scuwert et al. J. Biol.

Chem. 172: 221, 1948) and of.thrombin (Sherry

et al. J. Biol. Chem. 208: 95, 1954). Since, beside

other buffers, bicarbonate-Ringer’s solution serves
as medium, the degradation of BAEE can be taken
as the basis for a very sensitive manometric deter-
mination of trypsin. At pH 7.4 and 38°, the Q-value

(10-* m/hr/gm) is of the order of 10’, using 0.1-1.0

ug of crystalline trypsin per ml. BAEE also

undergoes hydrolysis in the presence of partially
purified bovine thrombin (Q = 3,000) and of

4 ave tae

AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS

391

venom of Vipera russelli (Q = 3,000) and of
several other snakes. With the help of this method
the specificity pattern of some proteases was in-
vestigated. Crystalline trypsin attacked not only
BAEE, but also benzoyl-.-histidine methyl ester.
The reaction rate (Q) was much lower than for
BAEE, namely 2 X 105. For thrombin and venom
of V. russelli, however, the difference of the rates
for both substrates was much smaller than for
trypsin. Results with the other substrates will be
submitted. Thus, in contrast to the generally
accepted view, trypsin and thrombin are able to
split not only derivatives of L-arginine, but of
L-histidine also. (Supported by a grant from The
Wander Co., Chicago, Ill.)

1279. Anthrone stability and determination of
the heptuloses. L. P. Zit. (introduced by
S. F. Carson). Biology Div., Oak Ridge Nail.
Lab., Oak Ridge, Tenn.

The formation of 5-(1,2-dihydroxyethyl)-2-
furfuraldehyde by acid treatment of p-altro-
heptulose (Z1Lu AND ToLBERT, J. Am. Chem. Soc.
76: 2929, 1954) suggested that the anthrone re-
agent should be applicable to the heptuloses.
However, the reaction of this p-altro-heptulose
with anthrone produces an absorption peak that is
partially obscured by a background absorbancy
of the reagent resulting from its widely recognized
instability. Nitrate ion was implicated in this
reaction. Addition of nitrates to a fresh reagent
results in an immediate darkening which is
identical in spectral properties with that observed
in aged reagents. This effect can be almost totally
circumvented by the combined use of low acid
(assaying less than 0.00002% nitrate ion) and
anthrone concentrations and by the addition of
thiourea as proposed by Rox (J. Biol. Chem. 212:
335, 1955). Under such conditions, quantitative
determination of the heptuloses is attained.

1280. Solubility transformation of a-lactal-
bumin. CuHar.es A. Zittie. Eastern Regional
Research Lab., Eastern Utilization Research
Branch, Agric. Research Service, U.S. Dept. of
Agriculture, Philadelphia, Pa.
a-Lactalbumin that is homogeneous by a variety

of physical tests is separated into 2 components

by a solvent-gradient extraction procedure.

The total preparation is precipitated by a con-

centration of 3.0m ammonium sulfate; the ‘soluble’

component (B) is extracted at 2.5 mM ammonium
sulfate concentration, and the ‘less soluble’ com-
ponent (A) requires 1.5 mM ammonium sulfate.

Solubility in 2 mM ammonium sulfate is used as a

simple test to distinguish the 2 a-lactalbumin

components: the ‘less soluble’ A is precipitated,
the ‘soluble’ B remains in solution. The ability of

392

a-lactalbumin to occur in these 2 forms appears
to depend on the binding of anions. In salt-free
solutions a-lactalbumin exists as preponderantly
(75%) component A. In the presence of 0.1 Mm
NaCl or a variety of other salts it is transformed
largely (89%) to component B. A and B do not
differ in the ultracentrifuge; in electrophoresis at
0.1 ionic strength a-lactalbumin is homogeneous.
A shift of the isoelectric point, determined by
precipitation and titration, from px 4.8 in salt-
free solutions to pH 3.6 in 0.5 m NaCl indicates

FEDERATION PROCEEDINGS

Volume 15

that the chloride anion and presumably other
anions, are strongly bound by a-lactalbumin,
The binding of anions and the apparent absence
of other physical changes suggests that the solu-
bility transformation is due to a change in physical
configuration implemented by the anion binding,
A study of precipitation in the isoelectric region
has shown also that 0.1 m NaCl salts-in thea-lactal-
bumin to some extent (about 1 mg/ml); this
effect is no longer apparent at 0.5 m NaCl con.
centration.

lume 1§

y other
Jbumin,
absence
he solu-
physical
binding,
¢ region
v-lactal-
1); this
Cl con.

AMERICAN SOCIETY FOR PHARMACOLOGY
AND EXPERIMENTAL THERAPEUTICS

Forty-sixth Annual Meeting

Attantic City, New Jersey, Aprit 16-20, 1956

An asterisk * following an author’s name indicates “by invitation”

1281. Vasopressin potentiation of morphine
action in rats. T. K. Apter, R. GEoreE* anp
H. W. Extrorr. Dept. of Pharmacology, Univ. of
California Med. Center, San Francisco.

The importance of antidiuretic influences on the
central depressant effects of morphine is suggested
by the fact that conditions leading to an increased
sensitivity to and/or concentration of antidiuretic
hormone result in an increased sensitivity to
morphine. Thus, adrenalectomy or dehydration
markedly enhances the central response to mor-
phine. Accordingly, the responses of rats to
morphine following an i.p. injection of 100 mu
vasopressin/kg were investigated. The LpDso of
150 mg morphine/kg for control male Long Evans
rats was reduced to 35 mg morphine/kg for vaso-
pressin treated rats; marked potentiation of the
‘analgetic’ response was also observed. The hy-
pothesis that this potentiation results from a
failure to excrete morphine was not substantiated
by results obtained 60 min. after intrapopliteal
injection of 2.0 mg/kg morphine-N-C“H;. Thus,
no difference was observed between control and
vasopressin-treated rats in the C!* data for ab-
sorption (94-99% dose), urinary excretion (40-
50% dose), plasma concentration (approx. 1.5
mg/l.), or brain concentration (approx. 0.06
mg/kg). In another experiment, rats were injected
subcutaneously with 8.0 mg morphine-N-C"H;/kg
and C'* concentration in brain and plasma de-
termined at 30, 60 and 150 min. after injection.
Employing an isotope dilution technique for de-
termination of free and total morphine, pre-
liminary results show no marked differences
between control and treated rats in plasma concen-
tration of free morphine. Total brain C' was
unaffected by vasopressin treatment. (Supported,
in part, by a grant from the Natl. Insts. of Health,
PHS.)

1282. Factors which affect biological activity
of thiopental. Kart AGre anp Cart ALPER

(introduced by J. R. DiPauma). Divs. of Pharma-
cology and Biological Chemistry, Hahnemann
Med. College, Philadelphia, Pa.

Factors which affect the biological activity of
thiopental are related to: 1) normal drug metabo-
lism, i.e. nutritional factors, 2) removal of free
thiopental from circulation, i.e. binding to serum
proteins and storage in depot fat. Levy, DiPalma
and Alper (J. Pharmacol. and Exper. Therap. 109:
377, 1953) described the test used to measure re-
sponses to thiopental. When wheat gluten, a poor
quality protein, replaces casein in the diet of mice,
they are considerably more sensitive to a PDw
dose of thiopental than casein controls; 1 of 25
climbs the plane in contrast to 27 of 48 in the
control group. When carbohydrate in the diet is
replaced isocalorically with lipid the mice are less
sensitive to a PDs dose of thiopental than the
casein controls; 19 of 27 climb the plane. The
results may be explained in terms of adequate
protein synthesis and the size of the body fat
compartment. It has been shown that plasma
proteins interact with thiopental, thereby af-
fecting an alteration in the biological activity of
this drug. These experiments on mice have shown
that thiopental in the presence of a 2% solution of
serum albumin is about 40% less effective than
thiopental alone when measured by the inclined
plane method. Similar experiments conducted on
the sleeping times of mice have demonstrated 40%
fewer mice slept 50% less time in the presence of
2% serum albumin. (Supported by Grant B-730
of the Natl. Insts. of Neurological Diseases and
Blindness, Natl. Insts. of Health, PHS.)

1283. Effect of flavonoids on vascular bed of
perfused rat hind limb. AntHony M. Am-
BROSE AND NicHo.as P. Piotnrxorr.* Pharma-
cology Section, Western Utilization Research
Branch, Agric. Research Service, U. S. Dept. of
Agriculture, Albany, Calif.

Various explanations for the mechanism of

393

ear

394

action of the vascular effects of flavonoids have
been advanced. In the present investigation the
vascular effects of the flavonoids—Rutin, narin-
gin, hesperidin methyl chalcone and _phos-
phorylated hesperidin—were studied in perfusion
experiments on rat hind-limbs. Pressure changes
registered by the perfusion fluid were used as the
criterion of alterations produced in the caliber of
vessels in the entire vascular bed. In preliminary
experiments determinations were made of 1) the
amount of each flavonoid required to produce a
definite effect, 2) the amount of epinephrine
necessary to cause constriction in the vascular
bed and 3) the amounts of adrenergic blocking
agents (dibenamine, priscoline) required to block
the epinephrine effect. When 10-20 mg of one of
the flavonoids was introduced into the perfusion
system together with epinephrine, the increase in
pressure (vasoconstriction) was invariably greater
than that produced by either agent alone. After
adrenergic blockade the flavonoids consistently
produced constriction while epinephrine did not.
The mechanism of action of flavonoids which
promote vasoconstriction has been ascribed to a
direct antioxidant action toward endogenous
epinephrine or to chelation with metals which
catalyze epinephrine oxidation. The present
studies suggest that, in addition to such actions,
the flavonoids, per se, have a vasoconstrictor
action, a suggestion in accord with the observa-
tions of Schiller (Am. J. Physiol. 165: 293, 1954).

1284. Pneumatic movement recorder for small
animals. (F. F. ANDERSON AND G. WAGLE
(introduced by A. J. PLuMMER). Research Dept.,
Ciba-Pharmaceutical Products, Inc., Summit,
N.J.

The apparatus to be described measures spon-
taneous movement of mice and has been used for
testing sedatives and stimulants. It is simple to
use and needs only infrequent cleaning to maintain
its proper working condition. The device is com-
posed of a spring-suspended cage attached to a
pneumatic movement amplifier which makes a
kymographic tracing through a spring-loaded
manometer. The manometer is kept on its base
line by air at constant pressure. A yoke suspended
over the open end of a T-tube set in the line
supports the cage; this yoke is arranged in such a
manner as to allow air to escape when the cage is
disturbed by animal movements. Escaping air
lowers the pressure and causes the manometer to
move. Mercury may be used as a by-pass to main-
tain a constant air pressure from the house
system; however, a specially designed valve is
more satisfactory. Great care must be taken in
setting up the apparatus as the adjustments are
quite critical. Uniformity in sizes and weights of
parts should be maintained throughout.

FEDERATION PROCEEDINGS

Vo ume ff

1285. Reversal of DL-penicillamine-cause
growth inhibition of rats by DL-serine,
H. VASKEN APOSHIAN AND BERNARD FRIEDLAND
introduced by B. H. Rossins). Dept. of Pharma
cology, Vanderbilt Univ. School of Medicine,
Nashville, Tenn.

Wilson and du Vigneaud (J. Biol. Chem. 184;
63, 1950) have shown that t-penicillamine cause
inhibition of the growth of young rats on a choline
free diet. They found that the inhibition wag
prevented by ethanolamine and related com.
pounds and that p-penicillamine was devoid of
growth inhibitory activity. Since serine is the
metabolic precursor of ethanolamine, experiments
were performed in our laboratory to ascertain
whether serine would prevent the growth inhi-
bition caused by t-penicillamine. After being
maintained on a choline free diet for 7 days, young
male albino rats were continued on this regimen
with the following supplements incorporated into
the diet: group I received 0.7% pu-penicillamine
hydrochloride; group II 0.7% pu-penicillamine
hydrochloride plus 0.4% pu-serine and group II]
0.7% vu-penicillamine hydrochloride plus 2%
pL-serine. After 18 days of supplementation,
group I gained 6 gm., group II gained 24 gm and
group III gained 49 gm. Studies are now in progress
to determine if L-penicillamine inhibits the con-
version of serine to ethanolamine.

1286. A diuretic screening procedure. Roy
AsTon* AND Harry W. Hays. Depts. of Pharma-
cology, Univ. of Toronto College of Medicine,
Toronto, Canada, and Wayne Univ. College of
Medicine, Detroit, Mich.

Rats were placed, in pairs, in metabolism cages,
allowing the collection of urine in graduated
cylinders and were given 2.5% body weight of
demineralized water by gavage. All pairs of rats,
whose total urinary excretion at the end of 2 hr,
was 40% or less of this primary hydrating dose,
were eliminated from the procedure. The re-
maining pairs were shown to be in a significantly
uniform state of hydration. These were given 5%
body weight isotonic saline by gavage concomitant
with the test drug in a volume of 1.0 cc. A com-
plete inhibition of the saline-induced oliguria ata
nontoxic dose was considered indicative of diuretic
activity. The effect of oral administration of
varying concentrations of sodium chloride (0.3-
2.0%) in the place of isotonic saline resulted in a
concave dose-response curve with its minimum
response at 1.0%. Mercuhydrin and Aminophylline
were investigated by this procedure and both
proved diuretic according to the criterion of the
method.

1287. Effect of adrenergic mediators on intra:
cellular distribution of potassium in the

oume I

-causel|
serine,
IEDLANI
Pharma

Ul edicine;

em. 184)
e cause
choline
‘ion was
ed com:
evoid of
e is the
eriments
scertain!
th inhi-
ar being
S, young
regimen
ited into
‘illamine
‘illamine
roup III
lus 2%
ntation,
gm and
progress
the con-

‘e. Roy
Pharma:
ledicine,
allege of

m cages,
aduated
eight of
of rats,
of 2 hr.
ng dose,
The re-
ificantly
iven 5%
omitant
A com-
Iria ata
diuretic
ition of
de (0.3-
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1) intra:
in the

March 1956

liver. G, V. AupIToRE* anp W. C. Houuanp.

Dept. of Pharmacology, Vanderbilt Univ. School

of Medicine, Nashville, Tenn.

During the past 2 yr. we have been investigating
the effects of several classes of drugs on the intra-
cellular distribution of potassium in various types
of tissues of the rat, cat, rabbit and guinea pig. It
was found that following the intravenous in-
jection of epinephrine and norepinephrine (80
y/kg) there was a transient fall in the K content
of the liver. Distribution studies showed that the
loss occurred primarily from the supernatant
fraction in the case of both drugs. Epinephrine
however had an additional effect not shown by
norepinephrine in that the former substance
causes a profound loss of potassium from the
mitochondrial fraction of the liver. The addition
of epinephrine (20-80 y/ml) to liver homogenates
also causes a loss of potassium from mitochondria
when these suspensions were incubated at several
different temperatures. Comparing the action of
epinephrine with that of the other classes of drugs
it was noted that in many respects epinephrine
produced effects similar to 2,4 dinitrophenol.
These findings suggested that epinephrine or one
of its metabolic products may be affecting certain
energy yielding reaction(s) in these particulate
components. Several oxidation products of
epinephrine were prepared, including adreno-
chrome, and were tested for their effects on the
intracellular distribution of potassium in the liver.
These experiments were equivocal. The intra-
venous injection of adrenochrome did not cause a
loss of potassium from whole liver tissue. How-
ever, in a number of experiments we observed
that this substance produced effects on the mito-
chondria similar to epinephrine.

1288. Inhibitory action of N-allylnormor-
phine on enzymatic demethylation of
narcotic drugs. JULIUS AXELROD AND JOSEPH
Cocuin. Nail. Inst. of Mental Health and Natl.
Inst. of Arthritis and Metabolic Diseases, PHS,
Bethesda, Md.

A TPNH-dependent microsomal enzyme system
which can N-demethylate morphine and other
phenanthrene analogues, methadone and meperi-
dine, has been described previously (J. Pharmacol.
& Exper. Therap. In press.) The effect of N-allyl-
normorphine on this enzyme system has been
studied. It was found that N-allylnormorphine (1
X 10-* m) reduced the enzymatic N-demethylation
of morphine and its analogues by 50%, meperidine
by 35% and methadone by 25%. The inhibition
was found to be in part competitive and in part
noncompetitive. N-Allylnormorphine (1 X 1074 m)
also inhibited the N-demethylation of pyribenza-

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

395

mine by 15% and the enzymatic conversion of
codeine to morphine by 40%, but had no effect on
the enzymatic N-demethylation of cocaine, the
side chain oxidation of hexobarbital and the de-
esterification of meperidine. The inhibitory action
of a number of normorphine analogues on the
enzymatic N-demethylation of morphine was also
examined. It was found that N-allyl and N-iso-
butyl substituted normorphine derivatives exerted
the greatest inhibitory action, while N-ethyl,
N-isopropyl and N-propyl. derivatives had the
least inhibitory effect. N-allylnormorphine can be
deallylated to normorphine by a microsomal
enzyme system. The enzyme systems which N-
demethylate narcotic drugs and the physiological
receptors to these drugs are similar in 2 respects:
they interact with the same substrates and are
antagonized by N-allylnormorphine. These obser-
vations would suggest that the enzyme systems
and the receptors have similar configurations.

1289. Inhibitory effect of local anesthetics
and their halogenated analogs on human
plasma and red cell cholinesterase. Nora
Baart,* SypNEy P. SHANOoR,* GERTRUDE R.
vAN Hees,* Ervin G. Erpés* anp Francis F.
FotpEs. Dept. of Anesthesiology, Mercy Hosp.,
and Section on Anesthesiology, Dept. of Surgery,
Univ. of Pittsburgh, Pittsburgh, Pa.

It has been previously reported (FouLpEs et al.
J. Am. Chem. Soc. 77: 5149, 1955) that halogen
substitution markedly increases the hydrolysis
rate of ester-type local anesthetic agents. In the
present study the inhibitory effect of procaine-
HCl (I), tetracaine-HCl (V), and 2-sec. butyl-
aminoethy]-4-aminobenzoate- HCl (VII) and their
2-chloro analogues (III, VI and VIII) as well as
the 2-fluoro (II) and 2-bromo (IV) analogues of
procaine: HCl on the hydrolysis of acetylcholine
by human plasma cholinesterase (PChE) and
human red cell cholinesterase (RChE) were
investigated by Warburg’s manomeiric method at
pH 7.4 and 37°C. The substrate concentration in
PChE was 2.2 X 10-2 mM and in RChE 3.0 X 10-* M.
The inhibitor concentration in the experiments
with PChE was 2 X 10-‘ m or 2 X 10° a. In the
experiments with RChE the inhibitor concentra-
tion was 2 X 10°‘ m. All the compounds had a
greater inhibitory effect on PChE than on RChE
and in general the inhibitory effect of the halogen-
substituted analogues was greater than that of the
parent compounds. Good correlation was observed
between the K,, of the compounds in plasma and
their inhibitory effect. There was no close correla-
tion between the enzymatic hydrolysis rate of the
various compounds by PChE and their inhibitory
effect. The I; values, calculated from the equation
V/Vi = 1+ K,/Ki X (I)/K, + (S), are:

396
Compound Iso QM)
PChE RChE

I 1.9%. 10° 2.8 X 10-4
II 1.8 X 10-* 1.1. 107"
III 2.7 X 10-5 3.5 X 10-5
IV 1.1 X 10-5 3.5 X 10-8
V 1.3 X 10-¢ 2.0 X 10-5
VI 1.6 X 10-* 4.8 X 10-°
VII 27 MAR? 2.8 X 10-4
VIII 8.6 X 10-5 8.5 X 10-5

1290. Effect of delayed treatment with plas-
min (fibrinolysin) on thrombi produced
with I!*!.labeled fibrinogen. N. Bacx,* J. L.
AmsBrus, J. W. Byron* anp S. SHULMAN.
(Dept. of Pharmacology, Univ. of Buffalo Med.
School, and Roswell Park Memorial Inst., Buffalo,
NTs
In previous reports a quantitative method has

been described for the in vivo testing of fibrinolytic
agents, based on determination of the decrease of
radioactivity over clots produced with ['#!-
labeled fibrinogen. Using this method a number of
proteolytic enzymes have been tested for fibrino-
lytic activity and pharmacologic properties.
Treatment was always started 30 min. after for-
mation of the clot. Of the enzymes tested, only
human and bovine plasmin (fibrinolysin) was
found to dissolve thrombi effectively in nontoxic
doses. In the present study venous thrombi were
formed with [!*!-labeled fibrinogen. Treatment
with plasmin was started at various time intervals
afterward. Histologic studies were undertaken on
the clots before and after treatment. Effectiveness
of therapy was evaluated in relation to the degree
of organization of the clot.

1291. Effect of mucopolysaccharides on the
lipemia clearing reaction. Davin H.
BakEpDER,* BERNARD SHORE,* JoHN W. GormMans*
AND JOSEPH SerrrTeR. Wyeth Inst. for Med.
Research, Radnor, Pa., and Donner Labs. for Med.
Physics, Univ. of California, Berkeley.

It has been reported (Proc. Soc. Exper. Biol. &
Med. 86: 709, 1954) that the injection of hyaluroni-
dase, partially degraded hyaluronic acid and DCA
caused the release into plasma of rats a factor
which was capable of clearing lipemic dog plasma
in vitro. In a continuation of these studies a series
of mucopolysaccharides closely related in struc-
ture to hyaluronic acid were screened by the
2-stage method for ability to cause the release of
a lipemia clearing factor. One of these compounds,
partially degraded mucin, was active orally and
was administered to humans. Two human volun-
teers each received 1.5 gm of partially degraded
mucin and one volunteer received 15 gm orally.
Blood samples were taken before and after treat-
ment and analyzed for change in ultracentrifuge

FEDERATION PROCEEDINGS

Volume 15

pattern, for release of free fatty acid from egg
yolk lipoprotein, and for the clearing of lipemic
dog plasma in vitro. Partially degraded mucin did
not have any effect on the ultracentrifuge patterns
of fasted patients, there was no release of free-
fatty acids, but there was optical clearing of
lipemic dog plasma. These findings suggest that
there may be 2 types of clearing, one which is the
result of a lipase in which the chylomicra are
reduced to free fatty acids, and the other a
physical dispersion of the chylomicra to smaller
lipid particles.

1292. Dibenzyline effect on hepatic ferritin
metabolism in relation to _ protection
against shock. Sitvio Barz,* §S. G. Sri-
KANTIA,* Irn1s ForBES* AND EPHRAIM SHORR.
Dept. of Medicine, Cornell Univ. Med. College,
New York City.

Further analysis of protective action of Di-
benzyline in hemorrhagic and traumatic (drum)
shock in the rat has revealed at least 2 mechanisms
through which this protection could be achieved.
One is through its adrenergic blocking action; by
blunting the extreme vasoconstrictive response to
hemorrhage or trauma, this agent insures a better
perfusion of the splanchnic viscera throughout the
shock syndrome and hence less anoxic damage
particularly to liver. A second mechanism,
namely a direct action on the intermediary
metabolism of ferritin within the liver, is now
reported. Brief (10 min.) aerobic exposure of
normal liver slices in vitro to Dibenzyline (2
ug/gm liver in 5 cc RPO,) has 2 metabolic effects:
a) it prevents the usual release of vasoactive
ferritin on subsequent anaerobic incubation and
b) it preserves the hepatic capacity to inactivate
ferritin aerobically after a 90-min. anaerobic
incubation, in contrast with the complete de-
terioration of this function in control liver slices.
Similar effects on hepatic ferritin formation and
inactivation are produced by Dibenzyline in vivo
in normal rats and those exposed to hemorrhagic
and traumatic shock. The importance of these
metabolic actions on the liver, in the protection
against shock afforded by Dibenzyline, is sug-
gested by experiments reported elsewhere on the
equally protective effect of a chemically related
agent, N-ethyl-N-hexahydrobenzyl-8-chloroethyl-
amine (G-D 131), which is devoid of adrenergic
blocking action but which exerts the same effect
as Dibenzyline on liver ferritin metabolism.

1293. Effects of depressants and convulsants
on the in vivo incorporation of inorganic
phosphate into acid-soluble nucleotides of
brain. James A. Barn. Dept. of Pharmacology,
Div. of Basic Health Sciences, Emory Univ.,
Emory University, Ga.

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March 1956

Intrathecal administration of IP** to mice has
been employed to study the incorporation of in-
organic phosphate into the acid-soluble nucleo-
tides of the brain. Within 7 min. after the ad-
ministration of the label all of the nucleotides in
the acid-soluble fraction which are resolvable
with a type I, Dowex-l-formate, ion exchange
column (HurxLBERT et al. J. Biol. Chem. 209: 23,
1954) show detectable labeling. ATP, GTP, UTP
and ADP are very strongly labeled. GDP, AMP,
DPN and several other unidentified guanine and
uridine derivatives show appreciable labeling.
The amount of label in ATP reaches a maximum
within 20-30 min. Depressants such as barbitu-
rates and chlorpromazine decrease, while con-
vulsants such as metrazol and thiosemicarbazide
increase the amount of in vivo labeling in most of
the nucleotides when measured during the early
time intervals. In general, the magnitudes of the
drug-induced changes are small and the results
are quite variable from experiment to experiment.
The results will be discussed in relation to previ-
ously reported in vitro data on drug effects in
phosphate transforming systems, and with respect
to the possible mechanism of action of these
central nervous system drugs.

1294. Effect of chlorpromazine on _ basal
ganglia electrical activity. W. W. Baker,
E. G. Szexety* anv E. A. Sprecex.* Dept. of
Pharmacology, Jefferson Med. College, and Dept.
of Exptl. Neurology, Temple Univ. School of
Medicine, Philadelphia, Penna.

In order to analyze the transient parkinsonism
observed sometimes during chlorpromazine medi-
cation, the effect of this drug on the electrical
activity of the caudate nucleus, the globus pallidus
and the sensory-motor cortex of the cat’s brain
was studied. Chlorpromazine in doses of 5-10
mg/kg injected intravenously produced slowing of
the electrical discharges of the striatum and the
pallidum. The amplitude of the discharges was in
most instances increased, in some the graphs were
flattened. The most striking change was the
appearance of spike discharges, single or in
groups, most frequently in the pallidum and more
rarely in the caudate nucleus. These changes
occurred within 5-10 min. and were only oc-
casionally accompanied by corresponding altera-
tions in the electrocorticogram. Various thalamic
nuclei (ventralis anterior, intralaminar nuclei,
centrum medianum, dorsomedial nucleus, etc.)
were stimulated bipolarly and the potentials
evoked in the striopallidum were recorded. These
evoked potentials, when present, were not ap-
preciably different from those elicited before the
application of the drug. It is inferred that this
increased pallidal activity could net be directly
linked to the action of the drug on the thalamic

evrary i

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

397

nuclei under study and that the action of chlor-
promazine is not limited to lower parts of the
brain stem but extends as far cranially as the
basal ganglia, particularly the globus pallidus.
(Aided by Grant no. B-470 of the Natl. Inst. of
Neurological Diseases and Blindness, Natl. Insts.
of Health, PHS.)

1295. Effect of colchicine on body tempera-
ture and urine volume in several species of
animals. R. W. Batex,* J. J. Kocsis* anp
E. M. K. Geruine. Dept. of Pharmacology,
Univ. of Chicago, Chicago, Ill.

Previous work has shown that colchicine
potentiated the effects of various hypnotics and
anesthetics in mice (J. Pharmacol. & Exper.
Therap. In press). Since depression of body
temperatures by low environmental temperatures
is known to prolong barbiturate-induced narcosis
in mice (Raventos. J. Pharmacol. & Exper.
Therap. 64: 355, 1938), the effect of colchicine in
depressing body temperatures was investigated.
In mice, colchicine (2 mg/kg) produced a 2°C
decrease in rectal temperature, but in rats and
hamsters depression of body temperatures and
potentiation of barbiturates occurred only with
supralethal doses of colchicine (8 mg/kg and 100
mg/kg, respectively). In guinea pigs, however,
potentiation of thiopental occurred with doses of
colchicine (0.4 mg/kg) which have no effect on
body temperature. Dehydration in mice is also
known to prolong barbiturate-induced hypnosis
(MANTHEI, personal communication). Since col-
chicine has been known to decrease the extra-
cellular fluid volume in rats (FERGUSON. Cancer
7: 607, 1954), the effect of colchicine on water
balance was investigated. In rats, colchicine
induced mild diuresis in the first 3 hr. after its
administration. This was followed by a period of
prolonged antidiuresis, the duration of which
appeared to depend on the dose of colchicine
administered. Antidiuresis was demonstrated in
animals that were given 5% of their body weight
(gavage) as water 3 hr. after colchicine. Anti-
diuresis was also seen in mice that were ad-
ministered colchicine but no initial diuresis was
noted.

1296. EEG-Flicker test for anticonvulsive
drugs. T. C. Barnes. Dept. of Pharmacology,
Hahnemann Med. College and Hosp. of Phila-
delphia, Philadelphia, Pa.

Previous experiments (Federation Proc. 13: 333,
1954; 14: 316, 1955) demonstrated that drugs used
in petit mal inhibit the electroencephalographic
response to flashing light in unanesthetized rabbits
in contrast to drugs used in grand mal which fail
to abolish the flicker effect. Mesantoin and bro-
mide have been found inactive against flicker.

398

Sedation was tested by tonic immobility, i.e. in
rabbit 225A the immobility lasted 3 min. which
increased to 58 min. 4 hr. after 0.7 mg/kg intra-
peritoneal potassium bromide. Mesantoin was
given by stomach tube in 1% tragacanth (usual
dose 200 mg/kg). Even 270 mg/kg mesantoin
(heavy sedation) did not abolish flicker but
protected against electroshock. The tonic im-
mobility test revealed postictal depression after
electroshock before the drug was given. Thus in
rabbit 235B immobility lasted 30 sec. which
increased to 2 min. following clonic convulsions
produced by 21 ma for 30 sec. Glutamic acid did
not abolish flicker nor electroshock. Glutamic
acid lactam did not affect flicker but protected
against electroshock. Thus rabbit 247B had a
clonic convulsion when 17 ma was passed between
mouth and occiput for 15 sec. but this was abol-
ished after 24 hr. i.p. 500 mg/kg glutamic acid
lactam. As previously reported milontin did not
inhibit flicker. Clinical reports state that milontin
is ineffective in classical 3/sec. spike-and-wave
petit mal. Subcortical pathways for flicker may
connect with the intralaminary system where
electrical stimulation produces petit mal seizures
in unanesthetized rabbits (MoNnniER. Helvet.
physiol. et pharmacol. acta 11: 73, 1953).

1297. Factors in activation of plasminogen by
streptokinase. JAMES W. BasTIAN AND R. G.
Hitt (introduced by N. Ercoui). Dept. of
Pharmacology, Armour Labs., Chicago, Ill.
Ratnoff (J. Clin. Investigation 32: 473, 1953) has

suggested that the activation of plasminogen by

streptokinase may be accelerated by the ‘process
of coagulation.’ We have studied this mechanism
in some detail and find that the fibrin substrate
itself appears to possess an accelerating function
in the activation reaction. Experiments indicate
that very little plasmin is formed during the
incubation of streptokinase and plasma (rabbit or
human) until a clot is produced. As fibrin is
formed the activation reaction apparently pro-
ceeds at a rapid rate for a short time (ca. 1-2 min.)
after which it is arrested, even though some
plasminogen-may still be present. The amount of
plasmin formed is related to the concentration of
streptokinase and complete activation is effected
at high levels. Our experiments suggest that the
partial activation may be explained on the basis
of a self-limiting mechanism whereby plasmin
acts back to digest streptokinase before total
plasminogen conversion can be accomplished. The
apparent need for fibrin in the activation of
plasminogen by streptokinase is a favorable
circumstance from the standpoint of therapeutic
clot dissolution. We obtained evidence indicating
that streptokinase, administered intravenously,
produces appreciable plasmin activity only at the

FEDERATION PROCEEDINGS

Volume 15

surface of an intravascular clot and does not
result in significant circulating protease activity.
The over-all result is that fibrin clots are spe-
cifically attacked in contrast with other proteins
found in the vascular system.

1298. Influence of antacids on _ salicylate
absorption and _ clinical effectiveness.
Rosert C, BATTERMAN, ARTHUR J. GROSSMAN*
AND EveLYN SomMer.* New York Med. College-
Metropolitan Med. Ctr., New York City.

The influence of antacids upon 1) rapidity of
absorption of aspirin from the gastrointestinal
tract of man and 2) clinical effectiveness and
intolerance of aspirin when utilized for therapy of
musculoskeletal conditions was studied according
to several plans. Groups of 10-15 subjects totalling
112 received 0.6 gm of aspirin with and without
buffered antacids. Free and tctal plasma salicylate
levels (method of Lester et al.) were determined
at intervals of 10, 15, 30, 60 and 120 min. The
presence of buffered antacids did not alter the
rapidity of absorption of salicylate from the
gastrointestinal tract nor the level of salicylate in
blood of man. A repeat study utilizing these
different methods for determination of blood
salicylate also failed to indicate any difference in
salicylate absorption. Alkalization with sodium
bicarbonate decreased the absorption of salicylate.
The average total plasma level at 10 min. de-
creased from 1.18 to 0.57 mg %. Although analgesic
effectiveness with chronic administration of
aspirin with and without buffers was identical,
intolerance to the buffered preparation increased
progressively from 13.5 to 45.0% of patients. The
data are at variance with the accepted tenet that
antacids favorably influence salicylate administra-
tion.

1299. Alterations in iron uptake as a function
of radiation dose rate. Srrgmunp J. Baum
AND Donatp J. Kime tporr (introduced by
R. W. Braver). Biological and Med. Sciences
Div., U. S. Naval Radiological Defense Lab.,
San Francisco, Calif.

The literature indicates that thé responses in
several biological systems to a given radiation dose
are influenced by dose rates below 10 r/min. The
present study reports the effects of dose rates
well below 10 r/min. (2 r/min., 1 r/min., 0.5 r/min.,
0.17 r/min.), upon a physiological system sub-
jected to 240 r of 1.3 mev gamma radiation.
Radioactive Fe®* incorporation into newly formed
erythrocytes was selected for the physiological
end point because of its high degree of radio-
sensitivity. Animals were injected at various
intervals after radiation exposure and the uptake
curves were determined from values obtained at
days 1, 2, 3 and 8 postinjection. Lowest Fe®®

jec
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ivity.

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ylate
ness.
MAN*
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ty of
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alling
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ylate
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March 1956

uptake was observed in animals injected with the
isotope 24 hr. postirradiation. The extent of this
depression was similar among the dose rate
groups. In rats injected at 48 hr. postirradiation,
significant differences in the pattern of uptake
were observed among the dose rate groups.
Increased iron incorporation was observed with
decreased dose rates. A moderate depression was
observed in animals injected at 72 hr. post-
irradiation and Fe®® uptake again varied inversely
with dose rate. Iron incorporation was normal in
the group injected at 120 hr. postirradiation. It is
concluded that dose rate influences inversely the
extent of iron uptake during the first 3 days post-
irradiation. It is possible that the magnitude of
damage observed in animals injected at 24 hr.
postirradiation masked the somewhat smaller
differences that can be ascribed to the influence
of dose rate at that time.

1300. Nephrotoxic serum nephritis; specificity
of the antigen. JAMES H. BaxTEeR AND Howarp
C. GoopMan.* Natl. Heart Inst., Natl. Insts. of
Health, PHS, Bethesda, Md.

The antigen in rat kidney tissue responsible for
the production of nephrotoxic (anti-rat-kidney)
antibodies in rabbits immunized with rat kidney
tissue has been regarded as relatively organ
specific, although evidence is available that the
antigen occurs to some extent in placenta and
certain other rat organs. We have found the
antigen in lung and placenta in amounts roughly
comparable to those in kidney and in many other
organs in smaller amounts. Not only are these
various organs capable of stimulating the pro-
duction of nephrotoxic antibodies, but they are
also able to absorb the nephrotoxic antibodies
produced by injections of kidney. Furthermore,
kidney is able to absorb the nephrotoxic anti-
bodies produced \y injections of lung. These
studies indicate that the nephrotoxic antigens in
various rat tissues are the same or closely related.
However, tissues from the dog are uot able to
absorb nephrotoxic antibodies produced by in-
jections of rat kidney. In addition, when dog
kidney is injected into rabbits, antibodies nephro-
toxic to the dog are produced but the antibodies
are without effect in the rat. Thus the antigen
responsible for the production of Masugi-type
nephritis apparently is species specific but not
organ specific. The observed preponderance of
renal injury following injections of antiorgan sera
presumably is due to: 1) occurrence of the antigen
in glomerular basement membranes which are
exposed directly to circulating antibodies, 2) the
relatively large amount of the antigen in the
kidney, 3) perhaps the ease with which renal
damage is detected and 4) perhaps the propensity
of renal damage for becoming chronic.

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

399

1301. Relationship of significance of wound
to pain experienced. Henry K. BEEcuHER.
Anesthesia Lab. of the Harvard Med. School at the
Massachusetts General Hosp., Boston, Mass.

The purpose of this study was to compare the
frequency and degree of pain (none, slight,
moderate, severe) in a situation 1) where the
wound has great advantage and means escape
from overpowering anxiety and fear of death on
the battlefield (war wounds terminating military
service) with a situation 2) where the wound
connotes disaster (major surgery in civil life). In
situation 1 it was found that extensive wounds
were associated with comparatively little pain; in
situation 2 lesser wounds were associated with far
more pain than in situation 1. The data are pre-
sented in quantitative terms. The essential
difference appears to be in the difference in anxiety
level in the 2 situations. Evidence of the im-
portance of anxiety in the production of pain is
presented. The observations indicate that extent
of wound bears only a slight relationship if any
(often none at all) to the pain experienced, and
support the 60-yr.-old concept of the importance
in suffering of the reaction component as opposed
to the original pain sensation.

1302. Tranquilizing effect of phenothiazines
in cats and rabbits. ALBERT J. BEGANy,*
JOSEPH SEIFTER, Harvey H. Puiess,* RicHarp
DEV. HuBER* AND WiLu1AM F. Bruce.* Wyeth
Inst. for Med. Research, Radnor, Pa.
Phenothiazine derivatives were compared with

chlorpromazine for ability to control mania

produced by morphine in cats and to produce
skeletal muscle relaxation and loss of awareness in

rabbits. Chlorpromazine and the related di-

methylamino, ethylmethylamino, diethylamino,

pyrrolidino and piperidino-n-propyl phenothia-
zines in doses of 5 mg/kg subcutaneously
administered 30 min. prior to morphine prevented
motor excitement but not pupillary dilatation nor
vomiting. The transient relaxation of the nicti-
tating membrane produced by morphine was
enhanced but duration was no greater than after
the phenothiazines alone. The same phenothia-
zines similarly administered at the height of
mania abolished motor activity but failed to
influence other manifestations of morphine
excitement. The above active compounds ad-
ministered to rabbits in doses of 25 mg/kg sub-
cutaneously resulted in miosis, lack of awareness,
poverty of movement, toleration of the back
position and variable loss of skeletal muscle tone,
the most marked resembling curare head drop.

The effects persisted in excess of 4 hr. Dimethyl-

amino-i-propyl, dimethylaminoethyl, diethyl-

amino and di-n-propylamino-n-propyl phenothia-
zines did not produce similar effects in rabbits.

400

Quaternization of active compounds abolished the
activity. Four of the compounds that were tested
in both cats and rabbits and found to be active
were also highly effective in controlling acute
agitation and hallucinosis in humans. (Chlor-
promozine kindly supplied by Smith, Kline and
French Labs.)

1303. Quantitation of analeptic activity in
pentobarbital depression. JuLIUS BELFORD
AND FrepericK F. Kao.* Dept. of Physiology
and Pharmacology, State Univ. of New York
College of Medicine at New York City, Brooklyn.
Analeptic action, as evidenced by changes in

total ventilation (BTPS), was studied in pento-

barbital-depressed dogs breathing 100% oxygen.

Increasing doses given intravenously produced

greater respiratory responses until a dose was

reached wherein effects began to diminish (lobe-
line, Metrazol, amphetamine and caffeine sod.
benz.) or nearly plateau (picrotoxin and Cora-
mine). At optimum dosage a statistically signifi-
cant response occurred at the first minute (6 min.
for picrotoxin) and this was also the time of peak
effect except for picrotoxin which showed a slowly
increasing action beyond an arbitrary 10-min.
observation period and which increase was usually
accompanied by tremors. In general, responses are

a function of the initial ventilation. Analysis

(optimum dose in mg/kg and first significant

percentage increase in ventilation) revealed that

Metrazol (6.25, 168%), while statistically equal to

lobeline (0.375, 328%) and amphetamine (2.5,

104%), was superior to a grester number of other

members of the series, i. :+affeine sod. benz.

(20.0, 70%), picrotoxin (0.60, 36%) and Coramine

(60.0, 29%). At a 7-10-min. period the analeptics

still showed significant increases; however,

amphetamine with an increase of 86% in ventila-
tion showed superiority in that, while statistically
it was equal to picrotoxin (64%), its effects were
greater than caffeine sod. benz., Metrazol, Cora-
mine and lobeline which displayed percentage
increases of 40, 38, 15 and 7, respectively. Picro-
toxin was equal in activity to caffeine and Metra-
zol and thése 3 were more potent than Coramine
and lobeline, with the latter now having the least
activity. (Supported in part by a research grant

(B-458(C2) from the Natl. Inst. of Neurological

Diseases and Blindness, Natl. Insts. of Health,

PHS.)

1304. Analgesic activity and toxicity of Ro
2-7113. W. M. Benson, D. J. CunNniINGHAM,*
D. L. Hane* anp S. Van WINKLE.* Dept. of
Pharmacology, Hoffmann-La Roche, Inc., Nutley,
N.J.

The analgesic activity and toxicity of Ro 2-7113

(1 - methyl - 3 - allyl - 4 - phenyl - 4 - propionoxy -

FEDERATION PROCEEDINGS

Volume 16

piperidine hydrochloride) were studied in several
animal species. This agent, the 3-allyl substituted
derivative of alphaprodine, was equally as toxi¢
but considerably more active than alphaprodine
hydrochloride and levorphan tartrate when ad-
ministered intravenously to mice. A high ratio
between the 50% lethal and analgetic doses was
evident also in mice when the agent was given
subcutaneously and. orally. Similarly, Ro 2-7113
exhibited a high order of analgetic activity in rats,
The ratio between toxicity and analgesia was
greater than that of alphaprodine and levorphan
when the drugs were given intravenously, subcu-
taneously and orally. In rabbits, comparable
results were obtained by the intravenous route of
administration although equivalent degrees of
analgesia were associated with similar degrees of
respiratory depression. Significant analgetic
effects were demonstrated with Ro 2-7113 in the
dog.

1305. Effect of meprobamate (Miltown) on
animal behavior (Motion picture). FRanK M,
BERGER, CHARLES D. HENDLEY AND Tuomas E,
Lynes.* Wallace Labs., New Brunswick, N. J.
2-Methyl-2-n-propyl-1,3-propanediol dicarba-

mate, called meprobamate (Miltown), is a tran-
quilizing agent used clinically for the relief of
anxiety and tension. In the rhesus monkey,
behavioral changes are seen different from those
caused by barbiturates, chlorpromazine or reser-
pine. The ordinarily hostile and fearful animal
becomes calm and relaxed, while retaining full
awareness of its environment and a normal
appetite. The animal loses its fear of the experi-
menter. Meprobamate is also a muscle relaxant
by virtue of its interneuronal blocking activity.
In cats, the hindlegs are affected to a greater
extent than the forelegs. An intraperitoneal dose
of 50 mg/kg causes loss of placing reflexes, paresis
of the hindlegs and ataxia, while the cat remains
alert and will attempt to catch mice. The effect
of meprobamate on electrical activity of the
cerebral cortex, caudate nucleus, hypothalamus
and nucleus ventralis lateralis of the thalamus are
shown. Specific effects of small doses of mepro-
bamate are seen only in the thalamus. At a dose
of 20 mg/kg, there is increase in amplitude and
slowing of frequency in the thalamus. The cortex
shows slowing only at much higher doses. This
observation may be correlated with the clinical
effectiveness of meprobamate in relieving anxiety
and tension without causing drowsiness.

1306. Correspondence between bacteriologic
responses of urinary infections to treat:
ment with nitrofurantoin and responses off
isolated bacteria to in vitro sensitivity
tests. Ernst H. Beutner, Howarp E. Lind

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E. Lind

March 1956

AND Howarp M. Trarron (introduced by
Perer B. Drews). Sias Labs., Brooks Hosp.,
Brookline, Mass.

A tentative selection of conditions and criteria
for in vitro bacterial sensitivity tests to nitro-
furantoin was made on the basis of our previous
study (BEuTNER et al. Antibiotics Annual, 1954-55,
988), using one group of cultural conditions. The
objectives of this study were to recheck the
tentative conclusions of the previous study and to
extend observations to other cultural conditions.
Direct comparisons between in vivo and in vitro
reactions were investigated. Ten new cases and 7
cases from the previous study were included. Four
disk concentrations on 3 media and 2 tube dilution
methods were evaluated. Results obtained with
the newly isolated organisms are in agreement
with findings of the previous study insofar as the
2 studies overlap. Correspondence between in
vivo and in vitro responses was obtained with 16 of
the 18 cultures tested by disk methods when the
optimum criteria were applied. Of 72 disk sensi-
tivity criteria tested, 10 yielded optimum results.
When tested by tube dilution methods, corre-
spondence was obtained with 15 of the 18 cultures,
applying optimal criteria. It is concluded that
100% correlation between in vivo and in vitro
responses to nitrofurantoin cannot be expected
but in vitro tests can be designed which are about
80-90% accurate.

1307. Comparative effects of some Rauwolfia
alkaloids on central vasoregulatory mecha-
nisms. K. P. BHarGcAva* anp H. L. Bortson.
Dept. of Pharmacology, Univ. of Utah College of
Medicine, Salt Lake City.

In view of our previous finding that reserpine
does not account for certain important anti-
hypertensive effects of Rauwolfia serpentina, it was
important to investigate under similar experi-
mental conditions other individual alkaloids
contained in this plant. The stereotaxic technique
was employed electrically to evoke pressor
responses from the medullary vasomotor center in
vagotomized cats on artificial ventilation. Spinal
vasopressor reactivity was determined by spinal
fluid compression after cord ligation at C7. Effects
of the alkaloids studied can be compared con-
veniently with those of reserpine (1-5 mg/kg i.v.)
and alseroxylon—a purified mixture of alkaloids
(1-5 mg/kg i.v.). Reserpine produced mild inhi-
bition of supraspinal excitability without sig-
nificant spinal depression, whereas alseroxylon not
only reduced supraspinal excitability but also
produced a primary depression of spinal pressor
reactivity. Serpentine (2-8 mg/kg i.v.), like
alseroxylon, manifested its predominant effect at
the spinal level and this was associated with a
significant fall in blood pressure. Ajmaline (2-5

eerrre i

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

401

mg/kg i.v.) also resembled alseroxylon; but the
effects of ajmaline, at both supraspinal and spinal
levels, were less pronounced than those of serpen-
tine; the effect of ajmaline on the blood pressure
was variable. Rescinnamine (2-5 mg/kg i.v.), like
reserpine, effected only subtle changes in central
excitability without producing a notable fall in
blood pressure. Reserpinine (5 mg/kg i.v.), except
for the fact that it produced a rise in blood pres-
sure, was similar to reserpine in its effects on
central vasomotor excitability. The pressor
response to norepinephrine was potentiated by all
the alkaloids mentioned. The inhibitory effects of
the individual alkaloids on the carotid occlusion
pressor response were not as clearly defined as
were the effects on central excitability. (Supported
by Grant no. B-491, Natl. Insts. of Health, PHS.)

1308. Assay of antidiuretic principle in
normal and long-term diabetes insipidus
dogs. WiLL1AM P. BLackmore (introduced by
ANDRES Gotu). Dept. of Pharmacology, Univ. of
Texas, Southwestern Med. School, Dallas.

It is generally considered that the secretion of
the antidiuretic principle by the posterior lobe of
the pituitary is dependent upon the anatomical
integrity of the supraoptico-hypophyseal tract,
the supraoptic and paraventricular nuclei and the
neurohypophysis. In addition, it has been reported
that the hypothalamus forms and releases anti-
diuretic principle after a functional reorganization
in the absence of the posterior lobe in experiments
on rats. In this study experiments were carried out
to determine the concentration of circulating
antidiuretic substance in dogs that had been in a
state of experimental diabetes insipidus for a
minimal period of 18 months. Assay for anti-
diuretic activity was performed on rats rendered
diuretic with alcohol and water. The presence of
circulating antidiuretic principle was assayed in
plasma obtained from both normal and diabetes
insipidus dogs. The concentration of antidiuretic
principle in the normal animals was a mean of
80 zu equivalents of vasopressin/ml of plasma. In
contrast, plasma from diabetes insipidus animals
showed no demonstrable circulating antidiuretic
principle. Therefore, since with this technique it
was possible to detect as little as 20 uu equivalents
of vasopressin it appears evident that the anti-
diuretic principle is negligible in these dogs. In
addition, urine production of these animals
maintained in a temperature controlled room
varied from 3000 to 5000 ml/day in contrast to
250 to 450 ml/day from normal dogs under similar
conditions, indicating additional evidence of
minimal antidiuretic principle. From these results
it would appear that the antidiuretic principle
released by the hypothalamus of long-term

402

diabetes insipidus dogs as measured by its concen-
tration in plasma was of an insignificant amount.

1309. Action of purines, nucleosides and
nucleotides on the guinea pig tracheal
train. HaroLtp BLuMBERG, Hyman B. DaytTon*
AND SAMUEL M. Gorpon.* Research Labs., Endo
Products, Inc., Richmond Hill, N. Y.

The action of certain purines, nucleosides and
nucleotides has been studied on the isolated
tracheal chain of the guinea pig. All of the com-
pounds either produced some degree of relaxation,
corresponding to tracheo- or bronchodilatation, or
were inactive; none produced contraction. Only
the adenine series caused noteworthy relaxation.
With reference to theophylline as 100, the approxi-
mate tracheodilator activities in terms of weight
and molar equivalents, respectively, were:
adenine 30 (25), adenosine 25 (35), 5-adenylic acid
25 (50), adenosine triphosphate 30 (85), and
2-aminoadenine 15 (12). Hypoxanthine and inosine
had slight activity (about 5). Guanine, guanosine
and guanylic acid had very little activity (about
3). Essentially no relaxation was produced by
xanthine and xanthosine, as well as by allantoin,
uric acid, 2-thiohypoxanthine, 8-azaguanine and
8-chloroxanthine. These findings correlated, in
general, with various literature reports on the
effect of purines, nucleosides and nucleotides on
other forms of smooth muscle, i.e., in vaso-
depressor and coronary dilator studies. One
notable difference was that tracheodilatation was
produced by adenine as the purine alone and did
not require the riboside form, adenosine, for
activity. Tracheodilatation and bronchodilatation
by purine forms alone are well known in the
methylxanthines. These gave the following
values: theophylline (1,3-dimethylxanthine) 100,
caffeine (1,3,7-trimethylxanthine (35, theobro-
mine (3,7-dimethylxanthine) 25, and EN-609
(7-chloroethyltheophylline) 200. Theophylline
relaxation was neither prevented nor counter-
acted by xanthine, xanthosine, hypoxanthine,
guanylic acid, etc., with 20- to 40-fold excesses of
the latter compounds.

1310. Mouse test for constipating action of
narcotics; morphine, dihydromorphinone,
and 14-hydroxydihydromorphinone (Nu-
morphan). Harotp BLUMBERG AND STEVEN
Carson.* Research Labs., Endo Products Inc.,
Richmond Hill, N. Y.

A test has been devised to compare the con-
stipating action of narcotics on the basis of
scybala counts in mice. Groups of 10 mice, in
wire-bottom cages, were used for each dosage
level. Food was removed before the test. Seybala
counting was started 10 min. after subcutaneous
injection of the drug, with counts recorded 30, 60,

FEDERATION PROCEEDINGS

Volume 1§

120 and 180 min. thereafter. The 3-hr. total wag
calculated as percentage of the count of a saline.
injected control group. Morphine sulfate gave a
straight-line dose response curve over a range of
2.5-20 mg/kg (as base). Morphine sulfate, dihy-
dromorphinone hydrochloride (Dilaudid Hydro-

chloride, Hymorphan Hydrochloride) and
14-hydroxy-dihydromorphinone hydrochloride
(Numorphan Hydrochloride) were then com-

pared at the respective analgesic ED values, as
determined by the Eddy and Leimbach hot-plate
method. These dosages were (as base): morphine
5.0, dihydromorphinone 0.83, and Numorphan
0.33 mg/kg, in the ratio of 15:2.5:1. At these
equivalent analgesic dosages Numorphan was
significantly less constipating than either mor-
phine or dihydromorphinone. For quantitation,
3-level bioassays were made at the following
dosages: morphine 5, 10 and 20; dihydromor-
phinone 0.5, 1 and 2, and Numorphan 0.5, 1 and 2
mg/kg (as base). By weight Numorphan was 7.5
times as constipating as morphine and 1.2 times
as constipating as dihydromorphinone. As men-
tioned, the analgesic potency of Numorphan was
15 times that of morphine and 2.5 times that of
dihydromorphinone. Therefore, at equivalent
analgesic dosages Numorphan was 50% as con-
stipating as morphine and 53% as constipating as
dihydromorphinone.

1311. Pharmacological effects of 5-hydroxy-
tryptophan, the precursor of serotonin,
DonaLp F. Boapanskt, HERBERT WEISSBACH
AND SIDNEY UDENFRIEND (introduced by
BERNARD B. BropieE). Lab. of Chemical Phar-
macology, Natl. Heart Inst., Natl. Insts. of
Health, Bethesda, Md.

Interpretation of the central pharmacological
effects of serotonin are difficult to evaluate be-
cause the quantities which cross the blood-brain
barrier after parenteral administration are too
small to permit chemical detection. The finding
that 5-hydroxytryptophan (5HTP) can enter
brain and maintain levels of serotonin provides 4
means of studying the central pharmacological
actions of serotonin. Although 5HTP, serotonin
and its metabolite 5-hydroxyindoleacetic acid
appear in the brain after 5HTP administration,
the observed pharmacological effects parallel the
levels of serotonin, indicating that 5HTP acts
through its conversion to serotonin. The effects
summarized below appear in dogs, rabbits, rats
and mice. In the latter 2 species iproniazid, an
inhibitor of serotonin metabolism, was also re-
quired to bring the levels of brain serotonin to 4
value sufficiently high to evoke marked central
activity. The effects of 5HTP are: generalized
skeletal muscle tremors, loss of placing reactions,
postural incoordination, lacrimation, salivation,

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March 1956

piloerection, increased heart rate, increased
gastrointestinal activity, increased depth and
frequency of respiration and forced expiration,
loss of response to visual stimuli, pupillary dila-
tion and loss of the light reflex. These actions of
excess serotonin resemble those observed by
Brodie, Pletscher, and Shore (J. Pharmacol. &
Exper. Therap. In press) when reserpine releases
serotonin in the presence of iproniazid. Here the
excitatory effects are ascribed to a high concen-
tration of free serotonin. The 5HTP presumably
produces large enough amounts of serotonin to
saturate its binding sites, thus resulting in a
high concentration of free amine. Similar effects
are also produced by the hallucinogen, lysergic
acid diethylamide. The tremors and changes in
heart rate and respiration produced by 5HTP,
probably through its conversion to serotonin, do
not occur in anesthetized animals indicating a
central site of action at least for these effects.

1312. Blocking action of dilantin upon the
pituitary-adrenal system. DeEsmonp OD.
BONNYCASTLE AND Arvip J. Brapiey.* Dept.
of Pharmacology, Yale School of Medicine, New
Haven, Conn.

We have shown that chronic dilantin adminis-
tration to nonepileptic patients led to a signifi-
cant decrease in the excretion of urinary
ketosteroids and, after an initial rise, to a de-
crease in urinary corticoids. Attempts to produce
some such effect upon the pituitary-adrenal sys-
tem of the rat were initiated. It was found that
with 100 mg/kg doses of dilantin given over short
or long periods of time, a depletion of adrenal
ascorbic acid following unilateral adrenalectomy,
epinephrine s.c., vasopressin i.v., etc. was
blocked. However, the adrenals of these treated
animals showed a response to massive doses of
ACTH (1 mg i.p.), but somewhat less than in the
case of the control animals. Intravenous adminis-
tration of ACTH in wg amounts clearly demon-
strated that in the chronically treated animals
there is a reduced responsiveness of the adrenal
to ACTH. Hypertrophy of the adrenals was
observed and was seen to be almost at a maximum
after 1 wk. of treatment.

1313. Reversible nephrotoxic effects of bi-
phenyl. Atsert N. Bootu,* AnTtHony M.
AMBROSE AND Fitoyp DeEps. Western Utiliza-
tion Research Branch, Agric. Research Service,
U.S. Dept. of Agriculture, Albany, Calif.

In connection with chronic toxicity studies on
orally administered biphenyl to rats, urine
samples were collected for studies on the meta-
bolic fate of biphenyl. It was noted that the
volume of urine increased markedly with increas-
ing dosage of biphenyl. On the 2 highest levels of

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

403

biphenyl intake (0.5 and 1.0% of the diet) some
urine samples gave a positive benzidine test for
blood. After standing overnight, large volumes of
solids settled out of the experimental urines in
proportion to the level of biphenyl fed. The addi-
tion of either sulfosalicylic acid or acetic acid to
fresh urine aliquots gave protein precipitates
which increased in volume with increasing dosage
of biphenyl. These effects were negligible at the
0.25% level and absent at 0.1%. It was found
that the urine volume and amount of protein
excreted was increased at least 4-fold by the in-
gestion of a diet containing 1.0% biphenyl as
compared with control values. Within 30 days
following removal of the biphenyl from the diet,
the urine volume and protein content were prac-
tically normal. Histopathological examination of
kidney sections from normal and experimental
rats revealed a good correlation with the urinary
effects in that during the period of diuresis and
proteinuria there was distinct tubular dilatation.
When the feeding of biphenyl had been discon-
tinued for 30 days, only small scars and a few
dilated tubules in several places were in evidence.

1314. Influence of dehydration on pento-
barbital sleeping time in mice. JosEPH F.
BorzELLEcCA AND R. W. MantuHer (introduced
by J. M. Coon). Dept. of Pharmacology, Jef-
ferson Med. College, Philadelphia, Pa.
Experiments were carried out to determine

how barbiturate anesthesia is affected by dehy-

dration in mice. Dehydration was induced by
depriving the mice of water but not food. Male

Swiss-Webster mice were injected intraperitone-

ally with 50 mg/kg of pentobarbital sodium in a

volume of 0.2 ml/20 gm body weight. Ten mice

were usually used for each group. Sleeping time
was the period between the loss and the return
of the righting reflex. Mean sleeping time for con-
trol animals was 60 min. After dehydration for

24 hr. the mean sleeping time was 41 min.; after

48 hr., 66 min.; after 72 hr., 82 min. Experiments

were conducted to determine whether the ele-

vated serum sodium in these animals was the
factor responsible for the altered sleeping time.

Administration of hypertonic NaCl resulted in a

significant increase in sleeping time proportional

to the concentration of the solution injected. To
determine whether this increased sleeping time
was due to the increased sodium or to the in-
creased tonicity, 1 gm/kg NaCl was administered
in increasing volumes 10 min. prior to the pento-
barbital. The increase in sleeping time was pro-
portional to the tonicity of the NaCl. The time
interval between injections of NaCl and pento-
barbital also influenced the sleeping time. Studies
with other hypertonic solutions indicated that
both the tonicity of the solution and its ab-

404

sorbability determine its ultimate influence on
sleeping time.

1315. Chronic toxicity of FD& C Orange No. 1.
ANNE R. Bourke,* ArTHUR A. NELSON AND QO.
GartH Firzuuaeu. Dir. of Pharmacology, Food
and Drug Admin., Washington, D. C.

Groups of 24 weanling rats, equally divided as
to sex, were fed FD &C Orange No. 1 at 2%, 1%,
0.5% and 0% in the diet. Since there were no
survivors beyond the 5th wk. at 2%, observations
are confined to 1%, 0.5% and 0% levels. Body
weights and food intake were recorded weekly for
2 yr., blood counts being taken 4 times. Gross
findings at 1% were marked increase of mortality,
enlargement of spleens, leucocytosis and anemia,
diarrhea and growth depression. At the 0.5%
level kidneys showed more chronic congestion
than did controls, and there was some splenic
enlargement. Microscopically, spleens showed
uniformly chronic congestion and less often
slight hyperplasia and increased pigmentation.
Kidneys showed nephritis of the type common
in older rats with no difference among the groups
except in incidence. Fourteen dogs were fed the
color at 4 levels ranging from 5 mg/kg/day by
capsule (about 0.02% of the diet) to 1.0% mixed
in the diet. The 3 dogs on the lowest level sur-
vived for 5 yr. without showing an effect. At
higher levels, 0.2% or more of the diet, effects
were variable, some dogs surviving for relatively
short periods and others showing little or no
effect for long periods. Pathological changes were
in general of a nonspecific type; some animals
were found dead with little to explain the death,
while others were emaciated and showed organ
changes chiefly of inanition.

1316. Hydrolipotropic shifts in certain tis-
sues of albino rats bearing Walker car-
ecinosarcoma 256. ELpon M. Boyp, M. E.
Murpocu,* E. M. Ketty* anp C. E. Boyp.*
Dept. of Pharmacology, Queen’s Univ., King-
ston, Canada.

Tissues were removed for lipid and water
analyses from 54 to 88 pairs of twin albino rats, 1
of each pair of twins inoculated and 1 not in-
oculated with Walker carcinosarcoma 256. The
tumor weights were 52+36% of host weight.
The following significant (P < 0.01) mean per-
centage losses of wet weight were found in tumor-
bearing rats: trachea-larynx 9, cervical lymph
nodes 21, submaxillary salivary glands 25, dia-
phragm 26 and thymus gland 43. Shifts in the
mean levels of lipids and water, calculated per
unit nonlipid dry weight and significant at P =
0.01 or less, in the tumor-bearing rats were ex-
pressed as a percentage of the mean level in the
nontumor-bearing twins. The water level was

FEDERATION PROCEEDINGS

Volume 16

increased as follows: diaphragm 41, thymus 34,
trachea-larynx 33, cervical lymph nodes 20, sub-
maxillary glands 16. Neutral fat levels were
significantly lowered in 3 tissues: diaphragm 40,
cervical lymph nodes 38, and trachea-larynx 31,
Total cholesterol levels were increased in all 5
tissues by 20-113%. Free cholesterol levels were
increased as follows: trachea-larynx 28, sub-
maxillary glands 29, cervical lymph nodes 30,
diaphragm 38 and thymus glands 65. Significant
increases in phospholipid levels were found in 2
tissues, namely trachea-larynx 19 and thymus
giand 35. Hydrolipotropic shifts in thymus gland
pyramided in animals bearing tumors of inter-
mediate size. Pyramidal shifts were not proven
in the other tissues. (Aided by a grant from the
Natl. Cancer Inst. of Canada.)

1317. Temperature effects on _ radiocolloid
uptake by isolated rat liver. RaLpu W,
Braver, GreorGE F. Leona,* Roy J. Houto-
WAY* AND GRANVILLE M. Moorg.* U.S. Naval
Radiological Defense Lab., San Francisco, Calif.
CrPO, colloid clearance by the isolated rat

liver is greatly decreased, at any given perfusion
rate, by lowering the perfusion temperature.
This effect is reversible. The general character of
the function relating perfusion rate and CrPQ,
extraction efficiency is not changed with changing
perfusion temperature in the range between 17
and 39°C. Comparison of extraction efficiencies
in preparations perfused at sufficiently high rates
to saturate the hepatic vascular bed allow cal-
culation of the temperature coefficient of the
uptake reaction; the resulting values lead to a
molar activation energy of approximately 15,600
cal. (Qio = 2) for the uptake of CrPO, colloid by
hepatic RE cells. Under these conditions it be-
comes possible to study the effect of decreased
uptake efficiency upon the histological distribu-
tion of a single radiocolloid under substantially
constant circulatory conditions. Radioautography
illustrates the effect of changing perfusion rate
or extraction efficiency upon CrPO, distribution
in the liver.

1318. Treatment of Tabun poisoning with
atropine and metrazol. Rospert V. Brown
AND LEA M Somers.* Chemical Corps Med.
Labs., Army Chemical Ctr., Md.

Tabun, dimethylamidoethoxyphosphoryl cy-
anide, is a potent anticholinesterase. Following
the intravenous injection of 0.8 mg/kg in the dog,
the expected signs of acetylcholine accumulation
set in rapidly. Respiration, briefly stimulated,
failed in 1-2 min. The administration of atropine,
1 mg/kg, intravenously, relieved most of the signs
of acetylcholine accumulation; the nicotinic
effects were not abolished; the central nervous

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system effects were only partially abolished. In
dogs anesthetized with 30 mg of Nembutal/kg,
respiratory paralysis was temporarily relieved
by atropine, but usually recurred promptly.
Additional atropine was ineffective in restoring
breathing. The administration of 50 mg. of Metra-
zol/kg, intravenously, promptly re-established
effective spontaneous respiration which lasted
at least 3 hr. in 4 of 6 dogs.

1319. Evaluation of factors enhancing cardiac
force during hypothermia. THEopoRE G.
Brown, JR.* AND MARION DEV. Cotten. Dept.
of Pharmacology, Med. College of South Caro-
lina, Charleston, and Dept. of Pharmacology,
Tulane Univ. School of Medicine, New Orleans,
Ta.

Preliminary experiments with intact dogs
demonstrated that the force of the individual
ventricular contractions, as measured with the
strain gauge arch, is increased moderately during
hypothermia (J. Pharmacol. & Exper. Therap.
110: 8, 1954). An increase in cardiac force also
has been observed in experiments with isolated
perfused rabbit hearts indicating that hypo-
thermia has a basic influence in increasing the
force of contraction. Similar observations have
been made with isolated frog hearts by Hadju
and O’Sullivan (Enzymologia 14: 182, 1950) and
with isolated cat papillary muscle by Trautwein
and Dudel (Arch. ges. Physiol. 260: 104, 1954).
Further experiments have been conducted with
intact anesthetized dogs to evaluate the degree
to which secondary factors may be responsible
for this increment in heart force. Measurements
of changes in initial diastolic length, using
Cushny levers, indicated that there was no sub-
stantial change in heart size during hypothermia.
Determinations of the levels of circulating epi-
nephrine and norepinephrine (RICHARDSON et al.)
showed that there was a distinct increase during
hypothermia in the amounts of these agents
present in plasma. This increase in circulating
catechol amines was sufficient to account for a
significant portion of the increase in cardiac force
in intact dogs during hypothermia. Total sym-
pathetic block, using the technique of Brewster
et al. (J. Pharmacol. & Exper. Therap. 171: 37,
1952), resulted in an enhancement of the increase
in cardiac force during hypothermia. The levels
of circulating epinephrine and norepinephrine
were increased during hypothermia in these latter
experiments, corresponding to the increases seen
in dogs without sympathetic block. Blood vis-
cosity increased substantially during hypo-
thermia but could not be related to the increase
in cardiac force since the blood pressure fell
progressively as the body temperature was
lowered. Marked decreases in respiratory tidal

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

405

volume reduced but did not prevent the increase
in cardiac force during hypothermia; large in-
creases in respiratory tidal volume enhanced
the increase in cardiac force.

1320. Mechanism of inhibitory action of
stibophen on glycolysis of Schistosoma
mansoni. ERNEST BUEDING AND Joan Mac-
Kinnon Mansovur.* Dept. of Pharmacology,
Louisiana State Univ. School of Medicine, New
Orleans, La.

Low concentrations of organic trivalent anti-
monials (stibophen, antimony potassium tartrate)
inhibit the activity of phosphofructokinase of
Schistosoma mansoni (MANSOUR AND BUEDING.
Brit. J. Pharmacol. 9: 459, 1954). The present
investigation is concerned with the problem of
whether and in what manner this effect is related
to the inhibition of glycolysis of schistosomes by
stibophen. When glucose and ATP were used as
substrates, addition of purified phosphofructo-
kinase of rabbit muscle to homogenates of schis-
tosomes markedly increased the rate of lactic
acid production and reversed the inhibition of
glycolysis produced by stibophen. Addition of
this preparation of mammalian phosphofructo-
kinase did not affect the production of lactic
acid from fructose-1,6-diphosphate (HDP) by
schistosome homogenates. These observations
indicated that the conversion of fructose-6-
phosphate to HDP is the rate-limiting reaction
of the glycolysis of schistosomes. It was found
that aldolase of S. mansoni has a high require-
ment for HDP; relatively slight reductions in
the concentration of this substrate below the
optimum resulted in a sharp decline of enzymatic
activity. Therefore, decreased formation of
HDP, due to inhibition of phosphofructokinase
by stibophen, could affect critically the activity
of the aldolase of the parasite and thereby inter-
fere with its rate of glycolysis. (Supported by
Public Health Service Grant No. E-668.)

1321. Pharmacologic studies on 8-(para-
aminobenzyl) caffeine hydrochloride as
blood pressure depressant. Raymonp M.
Buretson,* W. Epwarp O’MALLEY* - AND
Joun C. Krantz, Jr. Dept. of Pharmacology,
Univ. of Maryland School of Medicine, Balti-
more.
8-(para-aminobenzyl) caffeine hydrochloride

[1,3,7-trimethyl-8-(p-aminobenzyl) xanthine hy-

drochloride] (PABC) was selected for detailed

pharmacologic study from an extensive group of
8-substituted xanthine derivatives screened as
blood pressure reducing compounds. Vasodepres-
sor response of laboratory animals to PABC
were measured in anesthetized and nonanes-
thetized animals, in hypertensive dogs and in

406

normal and hypertensive humans, The compound
was active orally and intravenously. In anes-
thetized animals an intravenous dose of 2 ml/kg
of a 2% solution resulted in a prolonged fall in
average arterial blood pressure of about 30%.
Renal blood flow was not significantly altered
and coronary circulation was markedly increased
following the administration of PABC. Mech-
anism of action studies revealed that PABC
apparently acts directly upon smooth muscula-
ture to elicit relaxation of tonus. The LD5o in
rats was found to be 210 mg/kg. Toxicologic and
pathological studies showed the compound to be
relatively innocuous to the common laboratory
animal.

1322. Characterization of cholinesterases in
chick cells cultivated in vitro. ALAN BuRK-
HALTER,* R. M. FEATHERSTONE, MARION
JoNnEs* AND F. W. ScHuELER. Depts. of Phar-
macology and Bacteriology, College of Medicine,
State Univ. of Iowa, Iowa City.

In earlier studies, the presence of acetylcholine
(ACh) in the culture medium was shown to have
a profound influence on the level of cholinesterase
activity in 15-day chick embryo intestine cul-
tivated for 8 days in vitro in roller tubes. When
no ACh was added during incubation, the level
of enzyme activity fell to about 40% of that
initially present. ACh added during incubation
prevented this fall. Through the use of ACh,
acetyl-6-methylcholine (MECh), and _benzoyl-
choline (BZCh), the enzyme was tentatively
characterized as the specific acetylcholinesterase
(AChE). The present report deals with 1) further
studies in which the substrates mentioned earlier
plus propionyl choline (PRCh) and _ butyryl-
choline (BUCh) were used to provide a better
characterization of the enzyme, and 2) studies
in which observations were made on the influence
of each of these 5 substrates and of 2 other ACh
analogues on the cholinesterase activity of the
chick intestine cultivated in vitro. These other 2
compounds were §-dimethylaminoethyl acetate
hydrochloride (DIME) and methyl-6-trimethyl-
ammonium ‘propionate bromide (RC—the ‘re-
versed-carboxy ACh’). The substrate activity
pattern indicated that the enzyme in this em-
bryonic tissue cannot be identified well as being
predominantly either AChE or the nonspecific
type (ChE). Comparisons of 1) the enzyme(s)
with AChE and ChE and 2) the abilities of the
various derivatives in the culture medium to
affect the hydrolysis of acetylcholine or of them-
selves have been made.

1323. Conversion of D-glucuronolactone-6-
C“ and L-gulonolactone-1-C™ to L-ascor-
bic-1-C" acid in the rat. J. J. BuRNs, CAROLE

FEDERATION PROCEEDINGS

Volume 16

Evans* anp E. H. Mospacu.* New York Univ,

and Columbia Univ. Research Services, Gold-

water Memorial Hosp., New York, and Lab. of

Chemical Pharmacology, Natl. Heart Inst., Natl,

Insts. of Health, Bethesda, Md.

Further evidence has been found for inter-
mediates involved in the biosynthesis of L-as-
corbic acid from p-glucose. p-glucuronolactone-
6-C"4 and L-gulonolactone-1-C"* were administered
to normal and Chloretone-treated rats and L-
ascorbic acid was isolated from tissues and urine.
The over-all conversion of p-glucuronolactone to
ascorbic acid was 2% and of i-gulonolactone 8%,
The biosynthetic L-ascorbic acid was found to be
labeled predominantly in carbon-1 (about 85%
of total C') indicating that the intact carbon
chain of p-glucuronolactone and t-gulonolactone
can be utilized for L-ascorbie acid biosynthesis,
Neither compound was converted to L-ascorbic
acid in the guinea pig (percentage conversion
less than 7p the values obtained in rats). Despite
the inability of t-gulonolactone to serve as a
precursor for ascorbic acid in guinea pigs, it is
metabolized in this species to the same extent as
in rats—60% was converted to expired CQ,
within 5 hr. after the dose.

1324. Metabolic fate of thiobarbital. M1Lron
T. Busu anp Berry Lentz Gray.* Dept. of
Pharmacology, Vanderbilt Univ. School of Medi-
cine, Memphis, Tenn.

Some years ago we reported that in the dog
thiobarbital was not metabolized in appreciable
amount to barbital (Federation Proc. 1947, 313),
as judged by the crude technique then used. The
development in recent years of spectrophoto-
metric methods of determining barbiturates and
our recent acquisition of a DK spectrophotometer
have encouraged us to begin reinvestigation of
this and similar problems with refined techniques.
We have found that the absorption spectra of
thiobarbital and barbital are markedly different.
In the concentration range 1-10 yg/ml thio-
barbital gives 2 pronounced absorption bands
with peaks at 305 my and 255 my, in aqueous 0.02
M ethanolamine, pH 10.4. Acidification to pu 2.5
depresses these absorption bands only slightly,
but shifts them to 288 my and 238 mu. Analytical
differentiation of thiobarbital from barbital has
been readily accomplished by taking advantage
of the well-known fact that the absorption of
barbital at 239 my for px 10-11 disappears com-
pletely at px 2.5. By an isolation procedure re-
sembling that of Giotti and Maynert (J. Phar-
macol. 101: 296) we have found that, after an oral
dose of 200 mg in a man, about 15% of the thio-
barbital was present in the urine of the first 36
hr. The amount of barbital which could be pres-
ent is much less than this. Further refinement of

(E
EI
tre

inj
pre
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the
por
wh

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gan
asc
ing
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gan
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ume 16

Univ.
Gold-
ab. of
, Natl,

inter-
T L-as-
ctone-
istered
and L-
urine.
one to
ne 8%.
1 to be
it 85%
carbon
actone
thesis.
scorbice
version
Yespite
e as a
s, it is
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(1 LTON
ept. of
f Medi-

he dog
eciable
7, 313),
sd. The
»photo-
tes and
ometer
tion of
niques.
etra of
fferent.
1 thio-
bands
us 0.02
pH 2.5
lightly,
alytical
tal has
vantage
tion of
rs com-
lure re-
. Phar-
an oral
ne thio-
first 36
e pres:
ment of

March 1956

the purification procedure will be necessary in
order to identify an ‘impurity’ which adds a
considerable absorption to that attributable to
thiobarbital at 238 my and px 2.5. (Supported by
Research Grant RG3615 from the Div. of Re-
search Grants, Public Health Service.)

1325. Metabolic hydroxylation of 5-phenyl
derivatives of hydantoin. THomas C. But-
LER. Dept. of Pharmacology, Univ. of North
Carolina, Chapel Hill.
5-Ethyl-5-(p-hydroxyphenyl) hydantoin has

been found in the urine of dogs after the adminis-

tration of 5-ethyl-3-methyl-5-phenyl hydantoin

(Mesantoin) or 5-ethyl-5-phenyl hydantoin

(Nirvanol). The same product has been isolated

from the urine of a man receiving Mesantoin.

From urine of dogs and men receiving 5,5-di-

phenyl hydantoin there has been isolated 5-(p-

hydroxyphenyl)-5-phenyl hydantoin. The hy-

droxylated products as excreted in urine are
largely conjugated and can be released by acid
hydrolysis. Introduction of the hydroxyl group
in the phenyl ring results in a loss of the charac-
teristic pharmacological activity of the parent
compound. Products hydroxylated in positions
other than para have not been found. (Supported
in part by Grant B-384 from the Natl. Insts. of
Health, PHS.)

1326. Pharmacologic studies of pyrrolidyl-
methyl nortricyclene and bicycloheptenes.
ANNE CAMERON (introduced by Go Lv). John-
son & Johnson Research Fndn. and Research
Div. of Ethicon, Inc., New Brunswick, N. J.

A group of pyrrolidylmethyl nortricyclene
(ERL-235) and _ bicycloheptenes (ERL-145B,
ERL-240 and ERL-264), with quaternary ni-
trogens, were found to possess common phar-
macodynamic actions. Upon intravenous
injection, they caused an elevation of blood
pressure in anesthetized and spinal cats, with the
exception of ERL-240 which produced a depressor
effect. On the cat’s heart, perfused according to
the method of Langendorff, 0.5 mg of each com-
pound significantly reduced the coronary flow,
whereas the cardiac contraction and rate re-
mained practically unchanged. In the anesthe-
tized cat, they elicited contraction of the
nictitating membrane, dilatation of the pupil
and salivation (except ERL-240) upon intra-
carotid injection. By the use of ganglion-blocking
agents and/or removal of the superior cervical
ganglion in atropinized, anesthetized cats, it was
ascertained that the contraction of the nictitat-
ing membrane and the dilatation of the pupil
were due to stimulation of the superior cervical
ganglion. At higher doses in the anesthetized cat,
the contraction of the nictitating membrane was

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

407

more strongly inhibited following electrical
stimulation of the preganglionic fibers than
following stimulation of the postganglionic
fibers. This suggests a depression of the superior
cervical ganglion and also a depression peripheral
to the ganglion. All of these observations suggest
that these compounds share some of the actions
of nicotine.

1327. Failure of cortisone to inhibit acquired
immunity to Trypanosoma equiperdum in
the rat. Wiuu1AM CaNTRELL. Dept. of Phar-
macology, Univ. of Louisville School of Medicine,
Louisville, Ky.

Rats infected with Trypanosoma equiperdum
and cured with oxophenarsine acquired a rapidly
developing, highly specific immunity. When such
rats were challenged, 3-6 days after treatment,
by the intravenous injection of 10° trypanosomes/
kg, the trypanosomes disappeared within a few
minutes, while in nonimmune controls they could
be found immediately and increased progressively
until the death of the rat. The immunized rats
were not completely protected, however, and
after about 90 hr. were killed by a relapse strain
which differed antigenically from the original
population. We have used this system to test the
effect of cortisone on acquired immunity. Rats
treated with cortisone in doses up to 100 mg/
kg/day for 10 days beginning 2 days before the
initial infection and continuing 2 days after the
challenge injections did not suffer any impair-
ment of their ability to become immunized. These
results lead to the conclusion that the adverse
effects of cortisone on the curative action of
oxophenarsine, noted previously, were not due
to an impairment of acquired immunity. (Sup-
ported in part by a research grant (G 4117) from
the Natl. Microbiological Inst., Natl. Insts. of
Health, PHS.)

1328. Metabolism and enhancement of radio-
strontium excretion in man. Martin L.
CHARLES, Herta SpPeENcEeR, ARTHUR GOLDMAN,
SAMUEL KoRMAN AND DANIEL LASZLO (intro-
duced by Jut1an B. HerrMaAnn). Div. of Neo-
plastic Diseases, Montefiore Hosp., New York
City.

It has been previously reported from this
laboratory that radiostrontium metabolism can
be readily measured in man when strontium® is
administered as the tracer. The relationship
between calcium and strontium metabolism was
investigated and the enhancing effect of slow
infusions of calcium gluconate upon radiostron-
tium excretion was reported. Data will be pre-
sented which demonstrate that the metabolism of
strontium® differs greatly in patients, depending
upon their skeletal status, e.g. a patient with

408

excessively dense bones excretes in the urine
0.49% of the injected dose of strontium® in 24
hr. as compared to 20.1% by a patient with senile
osteoporosis. After 10 days the former excretes a
total of only 2.67% of the administered dose,
while the latter excretes 53.04%. The effects of
an anabolic hormone upon radiostrontium me-
tabolism will also be presented to illustrate that
strontium® may be used as a simple tool for the
study of bone metabolism and the effects of
therapy thereon. Further studies were carried out
on the enhancing effects of calcium on strontium®
excretion in terms of dose and time relationship
between exposure to radiostrontium and adminis-
tration of calcium.

1329. Influence of some CNS excitants on
tonic extensor component of maximal
seizures induced by pentamethylenetetra-
zol in mice. GRAHAM CHEN AND BARBARA
Bouner.* Research Labs., Parke, Davis & Co.,
Detroit, Mich.

The maximal seizures were induced in mice by
timed intravenous infusion of a 0.5% penta-
methylenetetrazol solution at the rate of 0.05
ec/10 sec. Ten mice were used to determine the
effect of each drug on seizure patterns 15-45 min.
after intraperitoneal administration. Agents
which were capable of abolishing the tonic-ex-
tensor component of the maximal seizures in all
animals at corresponding mg/kg dose levels are:
desoxyephedrine (20), d-amphetamine (40),
Meratran (20), Ritalin (120), cocaine (40), Bena-
dryl (20) and Ambodry] (40). It should be men-
tioned that the effects of the latter 2 compounds
are excitatory in mice. The following drugs were
ineffective at 40 mg/kg: procaine*HCl, ephedrine*
HCl, lysergic acid diethyl amide, morphine
sulfate, isonipecaine, methadone, hyoscine,
atropine, Artane and parpanit.

1330. Central depressant actions of ethyl
trichloramate [ethyl (2,2,2-trichloro-l-hy-
droxyethyl carbamate)|. GraHAM CHEN,
Joun W. Kisset* anp Epwarp P. Domino.
Research Labs., Parke, Davis & Co., Detroit,
and Dept. of Pharmacology, Univ. of Michigan,
Ann Arbor.

Ethyl trichloramate (HY 185) was found to be
equal to chloral hydrate in potency, onset and
duration of sedative and hypnotic effects when
given orally to rats. In mice it was more effective
than chloral hydrate in preventing the tonic-
extensor component of maximal seizures pro-
duced by strychnine, pentamethylenetetrazol or
electroshock. In acute high spinal transected cats
with bilateral vagotomy, HY 185 in doses of 10
to 25 mg/kg given intravenously in a 10% pro-
pylene glycol-water solution abolished the crossed

FEDERATION PROCEEDINGS

Volume 15

extensor reflex. Increasingly larger doses (up to
200 mg/kg) produced progressive depression of
the monosynaptic potellar reflex with a dose-
response curve similar to that of chloral hydrate,
HY 185 appears to be more selective in depressing
polysynaptic pathways in the spinal cord than
chloral hydrate; it is less selective than mephene-
sin.

1331. Analgesic-potentiating and _ diuretic
effects of 1-dimethylamino-3-cyano-3-
phenyl-4-methyl-hexane HCl (Z,) and
1 - dimethylamino - 2 - phenyl - 3 - methyl-
pentane HCI (Z;;,). James Y. P. Coen. Dept.
of Pharmacology, Marquette Univ. Med. School,
Milwaukee, Wis.

Utilizing a hot-plate method previously de-
scribed (Science 113: 631, 1951), subeffective
doses of morphine, 7 mg/kg and Z, or Ziss4, 10-20
mg/kg i.p. produced significant analgesia in mice
comparable to that induced by morphine, 20
mg/kg alone. Z compounds even in high doses.
showed no analgesia. Similarly, as determined by
a tail-pinching method using tygon-cushioned
Jacob’s Vulsellum forceps, subanalgesic doses of
morphine, 1-2 mg/kg and Z, or Zi34, 10-20 mg/kg
i.p. produced analgesia in rats, whereas with
morphine alone 4 mg/kg was required. Moreover,
these drugs exhibited remarkable diuretic ac-
tivity in rats as assayed by a simple device where-
by drops of urine from rats individually caged
atop § in. wire mesh were collected over a semi-
crimped, rapid absorption, 33 cm. diameter,
filter paper placed on similar mesh 1 in. below.
All rats were fasted overnight and given orally
1 ml/100 gm saline 1 hr. before dosing. The paper
under each rat was then changed every 30 min.
for a 6-hr. period and the urine-stained area
measured as an index of urinary output. The
mean (20 animals/dose) percentile increase over
control urinary output during the 5-hr. period
following respectively Zis,, 30 mg/kg i.m.; Z,, 30
mg/kg oral; Diamox, 20 mg/kg oral; Mercuhy-
drin, 8 mg (Hg)/kg im. and Mictine, 50 mg/kg
oral was approximately 375, 260, 310, 205 and 80%.
The oral and i.p. LDs0 of Zs, for rats was about
410 and 90 mg/kg; for mice, 440 and 190 mg/kg,
respectively. The oral LD5o of Z, for rats and mice
was approximately 370 and 510 mg/kg. Doses
above 60 mg/kg Z, or Z34 i.p. produced jumpiness
in these animals.

1332. Pharmacologic properties of N-2(3,4-
methylenedioxyphenylisopropyl) - nor-
epinephrine HCl (JB-251) and N-2(p-meth-
oxyphenylisopropyl) - norepinephrine HCl
(JB-245). James Y. P. Cuen. Dept. of Phar-
macology, Marquette Univ. Med. School, Mil-
waukee, Wis.

me 1§

up to
on of

dose-.
drate,
essing.

than
yhene-

iretic
no-3-

and
thyl-
Dept.
chool,

y de-
ective
10-20
| mice
ie, 20
doses.
ed by
ioned
ses of
ng/kg.
with
20ver,
Cc ac-
rhere-
caged
semi-
neter,
elow.
orally
paper
) min.
area,
_ The
- over
period
Z,, 30
cuhy-
ng/kg
80%.
about
g/kg,
| mice
Doses
piness

(3,4
nor-
neth-
HCl
Phar-
Mil-

March 1956

Using tracheal chains of guinea pigs (modified
method of*CastrLLo and DEBEER), JB-251 and
JB-245 exhibited approximately the same respira-
tory smooth muscle relaxant activity as Isuprel
(N-isopropylarterenol), the mean dosage of either
drug required to produce 100% relaxation of
histamine (2 ug/ml) induced tracheal contraction
being 0.01 ug/ml. As determined by the Anderson-
Craver apparatus using isolated rabbit hearts, at
the dose level of 0.0005 ug, JB-251, JB-245 and
Isuprel caused respectively an average 56, 50
and 62% increase in heart rate without significant
change in coronary flow. In lightly pentobar-
bitalized dogs, with 1 ug/kg i.v., JB-251 caused a
mean 31% increase in heart rate and 48% fall in
blood pressure which returned in a few minutes
to normal, increase in respiration and more pro-
longed decrease in intestinal tone. It was of in-
terest to note that when the dosage was increased
several thousand times (4 mg/kg, sometimes
even 10 mg/kg), the dogs still survived after
showing about 50% increase in heart rate, 92%
fall in blood pressure which usually returned to
normal in 2-3 hr., and a rather prolonged increase
in respiratory rate and decrease in intestinal
motility and tone. The oral and i.v. LDs0 of JB-
251 in rats was about 900 and 135 mg/kg; its i.v.
LD5o0 in dogs being approximately 10 mg/kg.
JB-245 showed about the same order of activity
as JB-251 but generally 4-6 and 6-10 times, re-
spectively, the oral and i.v. toxicity of the latter.

1333. Double blind comparison of Compound
22451, Pentothal and Surital. STantey M.
CHERNISH,* CHARLES M. GruBER, JR., MARION
DeMeyer,* SHIRLEY LITTLEFIELD* AND VIRGIL
K. Strorettine.* Lilly Lab. for Clin. Research,
and Depts. of Psychiatry and Anesthesiology,
Indiana Univ. School of Medicine, Indianapolis.
Sixteen psychotic, adult, female patients were

given a series of barbiturate anesthetics rapidly

intravenously prior to electroconvulsive therapy.

Placebos were also given to allow for evaluation

of postconvulsive anesthetic effects. The ADs5o’s

for Compound 22451, 1-methyl-5-allyl-5-(1-
methyl-2-pentynyl) barbiturate sodium salt;

Pentothal; and Surital were 22.3, 138 and 125

mg, respectively. The other significant findings

were as follows: Compound 22451 produced anes-
thesia more promptly than the thiobarbiturates;

administration of placebo was followed by a

significant rise in pulse rate, the barbiturates

were followed by a decrease; the duration of
postshock unconsciousness when 22451 was given
was intermediate, placebo was shortest and the
thiobarbiturates longest; the duration of ataxia
and apnea were shorter following placebo than
any anesthetic. Significant differences were not
observed between the patients receiving suc-

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

409

cinylcholine and those not receiving succinyl-
choline. No complications were produced in these
patients by these anesthetics.

1334. Modifications of pharmacology of re-
serpine and serotonin by iproniazid. Max
Cuessin,* BerNarRD Dusnick,* Epwarp R.
KRAMER* AND CHARLES C. Scott. Warner-
Chilcott Labs., Morris Plains, N. J.

It is known that iproniazid inhibits monoamine
oxidase, an enzyme which rapidly destroys
serotonin. It has been suggested that some ac-
tions of reserpine are mediated by release of
serotonin in the brain. Since the nature of action
of serotonin on the central nervous system is
unknown at present, it was felt that the use of
iproniazid might aid in elucidating the effect of
serotonin by potentiating the latter substance.
Studies were made in mice, dogs and cats, pre-
treated and posttreated with iproniazid in rela-
tion to doses of either reserpine or serotonin.
Results in mice showed that iproniazid caused a
marked reduction in mortality from reserpine.
Iproniazid-pretreated mice given reserpine (solu-
bilized in acetic acid) rather than being sedated
became restless, hyperactive and aggressive. This
reaction occurred within 30 min. following intra-
peritoneal administration of reserpine and only
5 min. after intravenous injection. The approxi-
mate duration of stimulation was 2} hr. Serotonin
given intravenously to iproniazid-pretreated
mice failed to cause excitation but intracerebral
serotonin did produce CNS stimulation. Dogs and
cats (anesthetized or unanesthetized) following
treatment with iproniazid responded to reserpine
with marked changes from the normal. The
iproniazid-altered responses to reserpine included
elevation of blood pressure, contraction of nic-
titating membrane, tachycardia, hyperpnea and
inhibition of myosis. Jn vitro studies of brain
homogenates from iproniazid-pretreated mice
demonstrated an inhibitory effect of serotonin on
endogenous oxygen uptake.

1335. Degradation of synthetic polymers into
potent estrogens. GrorGE P. CHIL. Roselle
Park, N. J:

Loosely cross-linked resins like polyacrylic
acid-divinylbenzene and _ polystyrene sulfonic
acid-divinylbenzene when fed to growing rats
produced very marked estrogenic effects, the in-
tensity of which were proportional to the dosage
of resin in the diet. At high dosages the testes
were of embryonic size and the females were in
continuous estrous. The estrogens were ex-
tractable with fat solvents but repeated tests
showed that there was a continuous degradation
of resin into estrogen. Although the highly cross-
linked resins such as used therapeutically for

410

salt removal were not estrogenic, at the dosages
fed to the rats, potent estrogens could be ex-
tracted from these resins in smaller amounts.
Complete conversion of these resins into estro-
genic substances was effected by pyrolysis. The
products obtained by pyrolysis were similar to
the fat solvent extracts. The estrogens were ac-
tive both orally and parenterally. The intensity
and duration of action depended upon the dosage
administered. The materials also induced a
granuloma at the site of injection. The granu-
lomas were transplantable simulating neoplastic
growth. A large dose 1-2 gm/kg in rats produced
anesthesia and respiratory depression. The
production of these active substances by the py-
rolysis of synthetic resins, suggestive of coal dis-
tillation, may open a new method for the de-
velopment of new drugs. In this particular case it
may also extend our knowledge relating to the
chemical configurations required for estrogenic
activity.

1336. Metabolism of S* chlorpromazine. JENS
CHRISTENSEN AND ARTHUR W. Wass (introduced
by JosepH R. DrPatma). Hahnemann Med.
College, Philadelphia, Pa.
$5 chlorpromazine (CPZ) has been prepared by

conventional methods and its purity determined

radiologically and pharmacologically. The dis-
tribution pattern has been studied in the tissues
of the rat and the mouse following various single
and multiple doses. Thirty minutes after the ad-
ministration of a single dose (8 mg/100 g) to mice,
the concentration of activity was in the following
descending order: brain, lung, heart, spleen, liver
and blood. Five days later all activities of the
tissues were greatly reduced, spleen and brain re-
taining the highest levels of activity. Excretion
into the urine was maximal at 12 hr. Examination

of the urine after single and multiple doses of S*

CPZ indicated the radioactivity to be in the forms

of free SO, “(3-5%), combined SO,” (23-48%) and

the sulfoxide of CPZ (49-73%). Activity was found
to accumulate in cerebral and hepatic tissue upon
repeated administration, most of the activity be-
ing found in the lipid fraction of these tissues.

Some activity was found fixed to hepatic proteins,

5-8% of total liver activity. Most of the activity

in the brain was located in the region of the hy-

pothalamus. Jn vitro studies showed CPZ to be
rapidly metabolized by brain, liver, kidney and
ileum homogenates at pH 7.2, 37°C and under
aerobic conditions. The principal products being
the sulfoxide and another component as yet uni-
dentified. (Supported by Smith, Kline & French
Labs.)

1337. Comparative effects of chlorpromazine
and reserpine on electroshock seizure

FEDERATION PROCEEDINGS

Volume 16

threshold. Howarp H. Curist1an,* Dorsry

EK. Houtrkamp, ArtHuR E. HEemMInG anp LEo

F. Mansor.* Research and Development Div.,

Smith, Kline and French Labs., Philadelphia, Pa.

Using the method described by Woodbury (Ree.
Prog. in Hormone Res. X: 65, 1954) as adapted for
use in our laboratories (Federation Proc. 11: 358,
1952), a study was made of the effects of chlorpro-
mazine hydrochloride (‘Thorazine,’ SKF No.
2601-A) and reserpine on electroshock seizure
threshold. Drugs were administered for 7-9 days
by single, daily, subcutaneous injections to both
intact and bilaterally adrenalectomized adult male
rats. Five or 6 rats weighing 200-250 gm were in-
cluded in each group. Adrenalectomized animals
were maintained on 1% NaCl as the drinking fluid.
SKF No. 2601-A was administered in doses of 10
and 20 mg/kg/day to intact rats and 10 mg/kg/day
to adrenalectomized rats. A 0.9% sodium chloride,
10% ethyl alcohol aqueous vehicle was used. Re-
serpine was administered in sesame oil at doses of
0.05, 0.5, 1.0, 3.0, and 6.0 mg/rat/day to both in-
tact and adenalectomized rats. The change in
threshold of each animal was determined with
reference to its own pre-treatment control. SKF
No. 2601-A had little or no effect on the threshold
in adrenalectomized rats, but did decrease the
threshold in intact rats. Reserpine, on the con-
trary, produced a lowering of threshold in both
adrenalectomized and intact animals. In the in-
tact animals, therefore, SKF No. 2601-A and
reserpine produced comparable effects. The dif-
ference in effect in adrenalectomized rats suggests
that their mode of action may be dissimilar.

1338. Partial purification of two malate oxi-
dizing enzymes in M. lysodeikticus. Davin
V. Coun (introduced by LAWRENCE PETERs).
Radioisotope Unit, VA Hosp., Kansas City,
Missouri, and Dept. of Pharmacology, Univ. of
Kansas Med. Ctr., Kansas City.

The oxidation of malic to oxalacetic acid in M.
lysodeikticus has been studied to characterize the
enzymes involved. Parallel assays for enzymatic
activity consisted of 1) spectrophotometric ob-
servation of the rate of oxidation of DPNH when
oxalacetate was the substrate and 2) manometric
determination of malate oxidation when ferri-
cyanide was employed as electron acceptor in
HCO;- buffer. The purification procedure included
the lysing of whole cells with lysozyme, sonic dis-
ruption of the insoluble residue and fractionation
with ammonium sulfate of solubilized material
freed by sonic treatment. In the course of purifica-
tion, a separation and 5-fold increase in specific
activity of 2 malic dehydrogenases was observed.
The first enzyme was approximately 150 times
more active in the manometric than in the spec-
trophotometric assay. The second enzyme was ap-

me 16

ORSEY
» LEo
Div.,
a, Pa.
(Ree,
ed for
|: 358,
orpro-
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eizure
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» both
t male
re in-
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g/day
loride,
d. Re-
ses of
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ige in
| with
. SKF
eshold
se the
e con-
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he in-
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ie dif-
ggests
a

e Oxi-
Davip
TERS).

City,
niv. of

‘in M.
ize the
ymatic
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March 1956

proximately 4 times more active when assayed
spectrophotometrically than when assayed mano-
metrically. The ferricyanide-reacting enzyme was
precipitated at a concentration of 20% ammonium
sulfate. It was atypical in its lack of requirement
for DPN (or TPN) in manometric studies and in
its low activity with DPNH and oxalacetate when
observed spectrophotometrically. Similarly, cya-
nide stimulated oxidation of malate 2-fold, but
was not essential for complete utilization of sub-
strate. That oxalacetate was the product of oxida-
tion of malate by this fraction was indicated by
theoretical oxygen consumption, R.Q., and quan-
titative oxalacetate formation. The DPNH-
reacting enzyme was precipitated in 30-40% am-
monium sulfate and resembled mammalian malic
dehydrogenase in the increased stimulation by
DPN after dialysis, the stimulation by hydrazine
and cyanide and the equilibria established between
oxalacetate, malate and DPNH. Studies concerned
with further purification of the 2 enzymes and
characterization of the oxalacetate produced by
the ferricyanide-reacting enzyme are in progress.

1339. Uptake distribution and excretion of
fission products in tissues of mice exposed
to a simulant of fall-out from nuclear
detonation. S. H. Coun, W. B. Lanu, J. K.
Gone, J. C. SHerwin, R. K. Fuser, L. L.
WILTSHIRE AND W. L. MILNE (introduced by
R. W. Braver). U.S. Naval Radiological Defense
Lab., San Francisco, Calif.

This study attempts to reproduce in the labora-
tory the acute exposure of animals to fall-out at
early times following nuclear detonation. The
ability of chemical agents to alter the uptake, dis-
tribution and excretion of fission products in mice
subjected to an acute inhalation exposure of a
fall-out simulant was investigated. The quantity
of fission products taken up by the animals as a
result of an acute exposure was proportional to
both the length of exposure and to the concentra-
tion of airborne radioactivity. The internally de-
posited activity was contributed chiefly by iso-
topes of short radiological and biological half life.
The longer-lived fission products were primarily
deposited in the skeletal system where they were
removed only very slowly. Although the radio-
logical decay of the activity in the various soft
tissues was quite similar at the early intervals fol-
lowing exposure, considerable variation occurred
in the rate of biological turnover of the material
in the tissues. The injection of zirconium citrate
prior to and just after exposure of animals to the
simulant resulted in a considerable decrease in
the concentration of the fission products deposited
in the skeleton and soft tissue. Similar treatment
with sodium EDTA resulted in no significant
changes in tissue distribution of residual activity.

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

411

The relatively small uptake of fission products and
rapid turnover in the tissues suggests that the
possibility of an internal radiation hazard occur-
ring following an acute inhalation exposure to
fall-out is very small.

1340. Release of free deoxypolynucleotide in
the spleen after systemic x-irradiation.
L. J. CoLE ann M. E. Exuis (introduced by R.
W. Braver). U. 8. Naval Radiological Defense
Lab., San Francisco, Calif.

Radiation-induced involution of radiosensitive
tissues, such as spleen, is accompanied by marked
losses of DNA from the tissue, and decreased DNA
concentration (L. J. Cote ann M. E. Ets,
Cancer Res. 14: 738, 1954). The early course of these
biochemical sequelae of ionizing radiations has
been investigated here as a possible basis for
elucidation of the mechanism involved. A scheme
of spleen analysis has been employed which makes
possible the partition and separate determination
of free DNA, i.e., deoxypolynucleotide, and DNA
present as deoxyribonucleoprotein (DNP). The
analytical procedure is based on the insolubility
of DNP in m/15 phosphate buffer at po 7.2, whereas
free deoxypolynucleotide is soluble in this medium.
The deoxypolynucleotide is further separated
from low molecular weight nucleotides by precipi-
tation with cold 0.2 m perchloric acid; the resul-
tant precipitate is washed with cold 0.2 m per-
chloric acid, extracted with hot 5% TCA, and its
DNA content determined by the Dische diphenyla-
mine procedure. In fresh buffer homogenates of
normal (nonirradiated) mouse spleen at least 98%
of the DNA content is present as insoluble nucleo-
protein; the soluble supernatant fraction obtained
after centrifugation of the homogenate contains
less than 50 wg polynucleotide DNA per 100 mg
fresh spleen. After lethal whole-body x-irradia-
tion (810 r), the deoxypolynucleotide content of
mouse spleen rises to 100 ug at 2 hr. The deoxypoly-
nucleotide level continues to increase with time,
attaining an average value of 450 ug at 6 hr. By
this time the spleen DNP content is approximately
50% of normal, and the ratio of deoxypolynucleo-
tide to DNP is 0.32. At 24 hr. postirradiation the
deoxypolynucleotide level is 20 ug. The data sug-
gest that loss of spleen DNA after x-irradiation
involves its release as soluble polynucleotide from
an insoluble, protein-bound form.

1341. Alleviation of toxic effects of veratrum
by chlorpromazine in treatment of hyper-
tension. Paut K. Conner, JR. AND Rosert G.
McConn (introduced by RusseLtt Huaerns).
Cardiac Clinic, Hermann Hosp., and Depts. of
Medicine and Pharmacology, Baylor Univ. Col-
lege of Medicine, Houston, Tex.

The narrow therapeutic-toxic margin of the

412

veratrum preparations has severely limited their
use in the long term treatment of primary hyper-
tension. Even though various investigators have
reported efficacious blood pressure response, the
emetic effect has prohibited extensive clinical use.
This study is an attempt to block this emetic ef-
fect of protoveratrine by the concomitant use of
chlorpromazine, utilizing its well described anti-
emetic properties. After control blood pressure
levels were established using placebo medication
in 24 mild to moderately severe hypertensive pa-
tients, a protoveratrine-reserpine combination was
begun and the dose was titrated in a step-wise
manner at weekly intervals until an adequate
blood pressure response was obtained or the emetic
dose was reached for each individual. Twelve of
the patients obtained a satisfactory blood pres-
sure response on the protoveratrine-reserpine
combination without nausea and vomiting. These
patients were followed without additional therapy.
The remainder of the group manifested the emetic
response before a significant blood pressure re-
sponse was obtained. The dose in these patients
was then readjusted to a level just below the
emetic level. Chlorpromazine was then begun and
was continued concomitantly with the protovera-
trine-reserpine combination and the dose of the
latter was again increased in an incremental fash-
ion. In about 2 of this group some reduction in
blood pressure was obtained without toxic effects
when the protoveratrine was increased to or to-
wards the previous emetic level. The remainder
of the patients tolerated protoveratrine dosages
in excess of the previous emetic level with addi-
tional blood pressure effect but without significant
toxic manifestations.

1342. Effect of fasting and alloxan on rat liver
desoxyribonucleic acid (DNA). EvuGENE
Conrap* AND ALLAN D. Bass. Dept. of Pharma-
cology, Vanderbilt Univ. School of Medicine,
Nashville, Tenn.

An increased DNA per unit weight of rat liver
following alloxan administration has been reported
by Diermeier, Bass and Di Stefano (J. Pharmacol.
& Exper. Therap. 107: 478, 1953). The animals were
subject to a fast for 2 days prior to alloxan injec-
tion. The present report considers the effect of
such fasting and postalloxan anorexia on rat liver
DNA and extends some previous observations
noted by Conrad and Bass (J. Pharmacol. &
Exper. Therap. 113: 11, 1955). The experimental
methods used are the same as were employed pre-
viously with the addition of microspectrophoto-
metric examination of pretreatment liver biopsies
and the use of pair-fed control animals. Two days
fasting induces a significant increase in the average
DNAP content of liver nuclei from 8.88 X 107!°

FEDERATION PROCEEDINGS

Volume 16

mg+0.37 to 10.8 X 10-!° mg+0.55 (P < .02). The
total number of nuclei per liver decreased from
28.2 & 108+1.1 to19.4 X 108+1.4 (P < .01). Micro-
spectrophotometrically, a shift to nuclei of higher
ploidy was noted. Thus, an increased DNAP con-
tent per average nucleus is observed prior to
alloxan treatment. The Feulgen method for DNA
evaluation reveals no significant difference be-
tween the mean values of the ploidy classes of pre-
alloxan liver biopsy samples and corresponding
specimens obtained at the time of killing. In the
pair feeding experiment, the alloxan treated rats
are not significantly different from corresponding
controls when the following are compared: liver
and body weights, total number of nuclei per liver,
total DNAP content of the liver, the amount of
DNAP per average liver nucleus and ploidy dis-
tribution. The above data suggest that the ele-
vated liver DNA observed following alloxan is
not due to the drug per se, but is the result of pre-
alloxan fasting and the self imposed limitation of
diet.

1343. Ganglionic stimulating and curariform
activity of dihydromurexine bromide (Ro
2-9101). Cecrt1a Conroy,* Beryt Kapper.t,*
Tina Frerruaera* anp LowEut O. RANDALL.
Dept. of Pharmacology, Hoffmann-La Roche Inc.,
Nutley, N. J.

Murexine is a ganglionic stimulant and curari-
form agent (V. ErspAMER AND F. Dorpont, Arch.
Internat. pharmacodyn. 74: 262, 1947). Dihydro-
murexine bromide (Ro 2-9101, [8-(4-Imidazolyl)
propiony]] choline bromide) is the reduced deriva-
tive of murexine bromide. Dihydromurexine is
about 4 times as strong as murexine in blocking
neuromuscular transmission in the cat, in raising
blood pressure in the cat and dog, and in stimulat-
ing the superior cervical ganglia in the cat. It is
less toxic in mice. Ganglionic stimulating effects
occur at the same dose level as the curariform ef-
fects in cats. The ganglionic stimulating effect in
cats is blocked by intra-arterial nicotine. The pres-
sor effect in dogs is blocked by hexamethonium.
The neuromuscular block is not antagonized by
edrophonium (Tensilon®) but is sensitized. The
compound is classified as a depolarizing agent at
ganglia and at the neuromuscular junction.

1344. Metabolism of D-4-amino-3-isoxazoli-
done (cycloserine) in man. GarLorp M.
CoNZELMAN, JR. (introduced by L. H. Scumrpt).
Christ Hosp. Inst. of Medical Research, Cin-
cinnatt, Ohio.

Previous studies from this laboratory (Antib.
and Chemo. 5: 444, 1955) have shown that of a
given dose of D-4-amino-3-isoxazolidone (cyclo-
serine) administered orally to rhesus monkeys, ap-

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March 1956

proximately 32% was excreted unchanged in the
urine within 72 hr. No nitroprusside-reacting sub-
stances other than parent drug were detected in
urine aliquots subjected to paper chromatography.
Similar analytical techniques have been applied to
urine samples collected from the human subject
after oral administration of cycloserine. Results
of these analyses indicate that no acetylcycloserine
was excreted and that the only urinary component
reacting with sodium nitroprusside had Rf values
identical with those of the parent drug. These
studies suggest that the apparent metabolism of
this drug by man is not unlike that observed in the
simian species. This preliminary study demon-
strates that in man, as in other animal species, an
appreciable fraction of the administered drug is
degraded to a substance(s) the characteristics of
which are still unknown.

1345. Antagonism by certain lactones of
digitoxin toxicity. GrorGE J. CosMIDEs,*
Tom S. Mrya* ann C. JELLEFF Carr. Dept. of
Pharmacology, School of Pharmacy, Purdue Univ.,
Lafayette, Ind.

The development of an effective antidote for
digitaloid toxicity was the objective of this inves-
tigation. Since the lactone moiety of the digitalis
glycosides is essential for cardiotonic activity,
other lactones may compete or displace the angel-
ica lactone of the digitaloid, or perhaps antago-
nize its toxicity by some other mechanism. The
pharmacodynamics of these lactones were also
studied. Screening for antagonism was accom-
plished by means of a chick embryo method in
which lethal dose responses were obtained with
digitoxin and with each lactone plus digitoxin.
Controls were studied simultaneously in each an-
tagonism experiment to preclude seasonal and
environmental variations which were observed.
Two modifications of the chick embryo method
were used. The drugs were administered topically
to the embryonic heart and complete cardiac ar-
rest was observed. In the second procedure the
embryos were pretreated by injecting the lactone
into the yolk 5 min. prior to the administration of
the Lp-99 of digitoxin. The same volume of saline
was injected into the yolk of the controls. The lat-
ter modification proved to be more sensitive. Five
of the 11 lactones tested demonstrated varying
degrees of antagonism. They were tetrahydro-
furfuryl alcohol, gamma-butyrolactone, nepetalac-
tone, glucuronolactone and gentiopicrin. Pharma-
codynamic studies were made in order to confirm
antagonism and further investigate these com-
pounds. Perfused frog hearts were used to deter-
mine threshold toxicity and subsequently eluci-
date antagonism either by alleviating arrythmias
or significantly prolonging the time for systolic

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

413

standstill. Lactone antagonism of digitoxin E.C.G.
changes in dogs were measured. Protection from
the acute toxicity of digitoxin in rats was investi-
gated.

1346. Relationship between stroke work and
ventricular contractile force. MARION DEV.
Cotten AND Harriet M. Mauna. Lab. of
Chemical Pharmacology, Natl. Heart Inst.,
Natl. Insts. of Health, Bethesda, Md.

The realtionship existing between stroke work
and ventricular contractile force was examined in
experiments with the heart-lung preparation
(HLP) of the dog. Pulmonary arterial, right atrial
and left atrial pressures were measured with
modified Jaeger water-mercury manometers and
aortic pressure was measured with a mercury
manometer. Aortic resistance was maintained ap-
proximately constant with a Starling resistance
and systemic output (cardiac output-coronary
blood flow) was determined with a Weese stro-
muhr. Ventricular contractile force was measured
with a modified Cushny lever equipped with a re-
sistance strain gauge coil (Am. J. Physiol. 172:
752, 1953). This latter system allowed the measure-
ment of ventricular contractile force at the initial
diastolic fiber length existing at the time of the
measurements. Stroke work and ventricular con-
tractile force were increased a) by progressively in-
creasing atrial pressures in graded increments and
b) by norepinephrine. The responses of both the
right and left ventricles were found to be funda-
mentally similar. Ventricular function curves for
the HLP were obtained when the atrial pressures
were raised in progressive increments up to levels
of 250 mm water. These function curves were
similar to those described for the intact dog by
Sarnoff (Physiol. Rev. 35: 107, 1955). A linear or
slightly curvi-linear relationship existed between
stroke work and ventricular contractile force in
the HLP. It is emphasized that each measurement
of contractile force was made at the initial di-
astolic fiber length existing at the time of the
measurement. Norepinephrine also substantially
increased both the stroke work and ventricular
contractile force. These parameters bore a linear
or slightly curvi-linear relationship to one another
as was the case in the experiments involving
increased atrial pressures.

1347. Technical factors that alter renal func-
tion in lumbar aortography. Crcit M.
Covuvss,* E. Stantey Crawrorp,* Joun H.
Moyer anp Micuart E. DeBaxey.* Depis. of
Medicine, Pharmacology, and Surgery, Baylor
Univ. College of Medicine, Houston, Tez.
Aortography has become a common diagnostic

procedure in the study of renal and vascular ab-

414

normalities. Complications following this pro-
cedure are rare, but occasionally acute renal
insufficiency is produced from which only about
60% of the patients recover. From clinical ex-
perience it would appear that several technical
factors play a role in the production of renal com-
plications. These are dosage of iodide, the site of
injection, and the rate of flow into the renal circu-
lation. The purpose of these experiments was to
study these factors in the dog. Seventy per cent
Urokon, sodium, the organic iodide preparation
commonly used clinically, was employed in the
experiments. Renal function before and after
aortography was determined by differential renal
studies. A dose of 0.35 ec/kg of 70% Urokon has
been found to be both safe and adequate for
aortography in the dog. No significant renal dis-
turbances followed the administration of this
amount of iodide into the open aorta. Upon in-
creasing the dose to 1.07 cc/kg severe renal damage
invariably followed. When 0.35 cc/kg of 70%
Urokon was injected into the aorta above the renal
arteries after occlusion of the aorta immediately
below these arteries, acute temporary renal in-
sufficiency ensued with recovery in 24 hr. Recovery
did not occur with larger doses. When the same
dose of Urokon was injected into the renal artery
acute persistent renal failure resulted.

1348. Cardiovascular effects of serotonin in
dogs premedicated with reserpine. GEORG
CRONHEIM AND JAMES T. Gourzis.* Research
Div., Riker Labs., Los Angeles, Calif.

Brodie et al. (Science 122: 968, 1955) have demon-
strated that in mice and rabbits reserpine releases
serotonin from its body depots (brain, intestines,
platelets). They postulated that the sedative ac-
tion of reserpine is produced through free sero-
tonin since they could obtain similar sedative ef-
fects by intravenous injections of serotonin. We
have attempted to correlate this release of sero-
tonin by reserpine to the cardiovascular effects
of the latter. Eight dogs, premedicated 24 hr. pre-
viously with 1 mg/kg reserpine intravenously, were
tested under urethane anesthesia before and dur-
ing infusion of serotonin creatinine sulfate (10
pvg/kg/min. in 0.5 ml/min.). Six dogs were bi-
laterally vagotomized. Serotonin infusion during
a 3-hr. period caused a gradual additional fall
in blood pressure and heart rate over and above the
reserpine-induced hypotension and bradycardia.
The blood pressure fell from 108+23 to 46+15 mm
Hg and heart rate in 6 vagotomized dogs fell from
101426 to 76416 beats/min. Integrity of the vagi
was not essential for these results. Serotonin infu-
sion did not cause further changes of the already
altered responses to afferent vagal faradization,
carotid occlusion or epinephrine. (Reserpine had
diminished the pressor responses to vagal stimu-

FEDERATION PROCEEDINGS

Volume 16

lation and carotid occlusion and had augmented
the epinephrine pressor response.) No effects of
serotonin infusion were observed in 5 control
animals. The results permit the interpretation
that the cardiovascular effects of reserpine, like
its sedative effects, may be related to free seroto-
nin in the brain.

1349. Applicability of new gas analyzer for
measuring respiratory gases. FRANK T.
CucniE, SAMUEL Kuna, MicHAEL KNIAZUK AND
F. RosBert PREDIGER (introduced by Hans
Mourtor). Merck Inst. for Therapeutic Research,
Rahway, N. J.

Kniazuk and Prediger have recently described
(Instrument Soc. of America, Sept. 1955, Paper
no. 55-9-2) the physical principles of a new sonic
gas analyzer for measuring oxygen consumption
and carbon dioxide output in respired air. Among
its advantages are high sensitivity (detects
changes of 0.01% oxygen concentration), operating
simplicity and stability and rapidity of determin-
ing changes in gas concentration (approximately
1 min. for oxygen consumption and 3 min. for
respiratory quotients). Our work confirms the
above-mentioned advantages and experiments
have been carried out in which the equipment has
been used in determining the effect of drugs such
as thyroxine, 2,4-dinitrophenol and Nembutal,
as well as other agents such as lysergic acid, re-
serpine and the cortical steroids on the basal
metabolism of rats.

1350. Anthelmintic activity of nicarbazin.
A. C. Cuckier, J. R. EGerton ann D. E. Foae
(introduced by Hans Mouttor). Merck Inst.
for Therapeutic Research and Chemical Div.,
Merck & Co., Inc., Rahway, N.J.

Studies on the anticoccidial agent, nicarbazin,
an equimolecular complex of 4,4’-dinitrocarbani-
lide and 2-hydroxy-4,6-dimethylpyrimidine, have
suggested that it also has anthelmintic activity.
Observations and reports from the field have con-
sistently indicated that infections of the large
roundworm (Ascaridia galli) are not a problem in
broiler flocks fed nicarbazin (0.0125%) in the ra-
tions. Experimental studies have been performed
to confirm these observations. Chickens were fed
nicarbazin and inoculated with 1000 embryonated
A. galli eggs and examined 3 or 7 wk. later. The
nicarbazin-fed chickens had fewer and smaller
worms than the controls or those fed rations con-
taining 0.0125% phenothiazine. The suppressive
effect of nicarbazin on the development of round-
worms was confirmed in another experiment. The
mean number of worms recovered from the nicar-
bazin-fed chickens was about 50% less and they
were smaller than in the controls. Studies with the
fowl cecal worm, Heterakis gallinae, showed that

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March 1966

nicarbazin was approximately as effective as
piperazine adipate. Swine fed 0.1% nicarbazin
were free from Ascaris lumbricoides, although
healed lesions characteristic of larval ascaris
migrations were found in the liver and lungs.
However, nicarbazin had no effect on the severity
of lung lesions from migrating ascaris larvae in
experimentally infected rats and mice. Nicarbazin
had slight anthelmintic effect on experimental
Schistosoma mansoni infections in mice but natural
infections of mouse pinworms were not affected.

1351. Synchronous recordings in the dog to
determine relations of the direct-body
ballistocardiogram with other cardiovascu-
lar measurements. T. D. Darpy* anp R. P.
Watton. Dept. of Pharmacology, Med. College
of South Carolina, Charleston.

Previous reports have described the procedure
of recording acceleration ballistocardiograms in
the dog and have characterized the changes oc-
curring with drugs such as arterenol, isopropyl
arterenol, phenylephrine and_ nitroglycerine
(DarBy, GOLDBERG, GAZES AND ARBEIT, Proc.
Soc. Exper. Biol. & Med. 86: 673, 1954; J. Pharma-
col. & Exper. Therap. 113: 14, 1955). These drugs
provide distinct and well understood variants in
the responses of heart force and diastolic pressure.
Continuation studies have provided synchronous,
high-speed recordings of the acceleration ballisto-
cardiogram, aortic pulse contours and directly
measured changes in heart force. The interpreta-
tions of Alexander (Federation Proc. 11: 738, 1952)
and Peterson (Federation Proc. 11: 762, 1952; Circ.
Research 2: 127, 1954) relating changes in the car-
diovascular forces occurring with systole to the
aortic pressure contours have served to explain
some of the more conspicuous changes in the con-
tour of the direct-body acceleration ballistocardi-
ograms. These ballistocardiographic changes were
considered chiefly in terms of the changes in the
slope of the systolic waves which exhibited a con-
sistent relation to the forces applied during the
ejection phase. An inverse relationship existed
between the amplitude of the systolic waves and
the forces lost to distention and friction in the
arterial system as interpreted from the contour
of the aortic pressure curves.

1352. Metabolism of glutethimide-C™. Bar-
BARA 8. D’Asaro,* HERBERT SHEPPARD* AND
A. J. PuumMer. Research Dept., Ciba Pharma-
ceutical Products, Inc., Summit, N. J.
Glutethimide (Doriden®), a-phenyl-a-ethyl-

glutarimide, labeled with C™ in the methylene

carbon of the ethyl side chain (prepared by H. and

K. Schmid of Zurich Univ.) was injected intra-

peritoneally into each of 10 male rats at a level of

100 mg/kg. Animals were killed by decapitation

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

415

at 1, 10, 120, 300 and 1200 min. after injection and
tissues and fluids analyzed for C'™ content. Un-
labeled glutethimide was added to the extracts
and then submitted to paper chromatography
using a 1:1 mixture of methanol and petroleum
ether as the developing solvent. Identification of
the glutethimide and derivatives retaining the
imide group was made by spraying the dried
chromatogram with alkaiine hydroxylamine fol-
lowed by acid ferric chloride 30 min. later. Radio-
active components were detected by radioautog-
raphy. The C content of the various tissues and
fluids as a function of time after the intraperi-
toneal injection of glutethimide-C™ will be dis-
cussed in relationship to the observed pharma-
cological activity.

1353. Effect of strrctural changes on the en-
zyme kinetics of procaine hydrolysis by
plasma cholinesterase. Davin L. Davis,*
Francis F. Fouprs, Vicror P. WHITTAKER.*
Dept. of Anesthesiology, Mercy Hosp., and Sec-
tion on Anesthesiology, Dept. of Surgery, Univ.
of Pittsburgh, Pittsburgh, Pa., and Dept. of
Physiology, Univ. of Cincinnati, College of Medi-
cine, Cincinnati, Ohio.

The maximum velocity (Vm) and the Michaelis
constant (Km) of procaine.HCl (proc.) ; N-diethyl-
aminoethyl-p-aminobenzoate methobromide
(PDMI-667); N-diethylaminoethylbenzoate. HCl
(PDMI-666) ; N-diethylaminoethylbenzoate meth-
iodide (PDMI-669) and benzoylcholine chloride
(BzCh) were determined in a Beckman DU
spectrophotometer using Kalow’s method (J.
Pharmacol. & Exper. Therap. 104: 122, 1952).
The initial substrate concentrations were 5 X 1075
M, the source of enzyme was purified human plasma
cholinesterase (Cholase), and its final dilution
ranged from 1:750 to 1:30,000 depending upon the
Vm of the compound investigated. The observed
Vm and Ky values (calculated according to LInE-
WEAVER AND Burk, J. Am. Chem. Soc. 56: 658,
1934) were as follows:

Compound Km (mM) Vm (m/l. x min.) Relative Vm
Proc. 2.6 X 10-* 3.6 X 10° 100
PDMI-667 10X10°* 4.7 X 10° 130
PDMI-669 3.5 10-* 1.6 10"! 4,450
PDMI-666 43x 10° 1.1X 10" 3,060
BzCh 2.2X 10% 1.2X 10 3,340

The findings indicate that the removal of the p-
amino group markedly, and quaternization of the
tertiary amino group slightly, increases the rate
of hydrolysis of the parent compound, proc.

1354. Arterial pressure and renal functions
during intravenous infusions of serotonin
in rats. FRANCESCO pEL Greco,* G. M. C.

416

Masson* anp A. C. Corcoran. Research Div.,

Cleveland Clinic Fndn., Cleveland, Ohio.

Erspamer (1952) found that subcutaneous in-
jections of about 4 ug/kg of serotonin (5-hydroxy-
tryptamine, 5-HT) in hydrated rats, decreased
urine flow (V) and, at larger doses, glomerular
filtration (GFR). We (1954) found antidiuresis in
rats given larger doses, but, in dogs given 5-HT
by intravenous infusion, found only moderate
hypotension at doses of 2-6 ug/kg/min. Effects of
intravenous infusions of 5-HT were therefore
studied in 3 dosage groups of anesthetized rats
during mannitol diuresis. The dosages were re-
spectively 1), 2-6, 2) 8-12 and 3) 13-19 ug/kg/min.
Functions studied were V, urinary osmolarity
(Uosm) and chloride (Uc), carotid arterial pressure
(BP) and, in some experiments, GFR. Results,
by groups, were as follows: 1) slight, tran-
sient decreases of BP only; 2) initial larger de-
creases in BP with recovery, but persistent
decreases in V, Uosm and Uc which, particularly
as regards Uosm and Uc, tended to persist after
discontinuing 5-HT administration; 3) biphasic
BP curve with initial fall and return to levels
higher than control; severe depression of renal
functions occurred and persisted in some animals
in association with macroscopic renal infarcts.
GFR was depressed in groups 2 and 8, more so in
the latter. The antidiuretic effects of 5-HT, seem
to depend on decreased GFR but the necessary
intravenous dosages are higher than those reported
effective subcutaneously. The persistent renal
defect after large doses is apparently due to is-
chemia and probably underlies the cortical necro-
sis it elicits (E. Pacg, 1955).

1355. Effects of chlorpromazine on E.E.G.
and its activation. EpmMunp W. J. DEMAaRr
AND W. R. MartIn (introudced by K. R. Unna).
Dept. of Pharmacology, Univ. of Illinois, College
of Medicine, Chicago.

The effects of chlorpromazine on the E.E.G. and
on its activation by auditory stimulation and by
epinephrine were studied in 40 unanesthetized
cats whose spinal cords were transected at C 1.
In 36 of these preparations chlorpromazine in
doses of 1, 5 or 10 mg/kg produced a diminution of
low amplitude high frequency activity. The result-
ing records of slow activity contained 1-3 cycles
per sec. waves, 8-12 cycles per sec. spindles and
7-10 cycles per sec. waves, that have a slower as-
cending phase than the sleep spindles. In some
preparations a transient period of activation of
1-2 min. duration was seen within the first 10
min. following injection of chlorpromazine. The
decrease in frequency and the degree of spindling
did not appear to be correlated to the dose. In
30% of the preparations chlorpromazine failed to
suppress the activation of the E.E.G. by auditory

FEDERATION PROCEEDINGS

Volume 16

stimulation. Activation of the E.E.G. was ob-
tained in 50% of the preparations by epinephrine
in doses from .5 to 3 ng/kg. With repeated injec-
tions of epinephrine the activation became in-
creasingly feeble and the injections were followed
by increasing appearance of spindles. In 25% of
our preparations chlorpromazine failed to block
the activation by epinephrine. Administration of
nor-epinephrine in doses from .5 to 3 ug/kg did
not produce activation. After repeated adminis-
trations it causes slowing of the E.E.G. and the
appearance of 8-12 cycles per sec. spindles. These
effects of norepinephrine become more pronounced
following the administration of chlorpromazine.
(This study was supported in part by research
Grant 983 of the PHS.)

1356. Possible role of serotonin on the hypo-
tensive and hypothermic actions of reser-
pine. Q. Dremrine,* Donatp F. Boepansx1,*
SipNEY UpENFRIEND, P. A. SHoRE* anD BER-
NARD B. BroprEe. Columbia Research Service,
Goldwater Memorial Hosp., New York City and
Lab. of Chemical Pharmacology, Natl. Heart
Inst., Bethesda, Md.

Previous reports have shown that a few hours
after reserpine administration to rabbits the drug
is no longer detectable in brain, whereas tran-
quillization and the block of the serotonin binding
sites persist for 48 hr. or more. These data sup-
ported the concept that the sedative effect of
reserpine is mediated through the action of free
serotonin. The report to be presented shows that
other central actions of reserpine persist long
after the drug can no longer be detected in the
brain. Following the administration of reserpine
(1-5 mg/kg) to unanesthetized rabbits, the hypo-
tensive effects persist 2 or more days. Other ef-
fects, some of which may be central, are also of
long duration. These include lacrimation, relaxa-
tion of the nictitating membrane, bradycardia,
and diarrhea. In unanesthetized mice that re-
ceived 1 mg/kg reserpine, body temperature fell
about 3°C for a period of 24 hr. and was still sub-
normal after 48 hr. Thus, certain central actions
of reserpine, such as lowering of blood pressure
and of body temperature, appear to be related
temporally to the concentration of brain serotonin
and not to the concentration of reserpine. This is
evidence that these actions of reserpine may be
mediated through serotonin and suggests the pos-
sibility that serotonin may have a role in regula-
tion of blood pressure and temperature.

1357. Effects of STH alone and in combina-
tion with ACTH and cortisone acetate on
EST. Satvatore J. DeSatva Anp R. A. Evans
(introduced by N. Ercour). Dept. of Pharma-
cology, Armour Labs., Chicago, Til.

» 15

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March 1966

We have studied in the past the adrenocortico-
pituitary ‘hormonal influences upon CNS sensi-
tivity. A difficulty in interpreting the findings is
the influence of body weight which per se affects
the electroshock threshold (EST). At this time we
have accumulated sufficient data to express the
threshold variation in relation to the figure ex-
pected on the basis of body weight. We present in
this form the effects of somatrophic hormone
(STH) alone and in combination with ACTH and
cortisone acetate in intact and hypophysectomized
rats. With 5 treatments per wk. of STH (0.5 mg
s.c.) in the nonoperated rat, no significant change
appeared during the first 6 wk., but afterwards,
the threshold was significantly increased, which
effect disappeared if treatment was interrupted.
In contrast, STH + cortisone acetate (0.5 mg
s.c.) did not produce a rise in threshold, which is
in agreement with the threshold lowering effect
of cortisone acetate previously reported. If this
combined treatment is stopped after the 6th wk.,
the EST is enhanced indicating a residual effect.
Continuous combined STH + ACTH (0.75 vu i.p.)
resulted in a threshold rise which disappeared by
stopping treatment. STH alone, or in combination
with ACTH or cortisone, did not increase the
threshold in the hypophysectomized rats. In fact,
during the early hyperexcitable phase, it decreased
the EST. Blood sugar determinations in 20 hr.
fasted animals (intact and operated) showed sur-
prisingly hypoglycemia in most instances of in-
creased EST.

1358. Tissue damage by macromolecules. L. E.
Derricx,* H.C. UpHam* anp THomas J. HALEY.
Div. of Pharmacology and Toxicology, Atomic
Energy Project, School of Medicine, Univ. of
California, Los Angeles.

Rabbits with indwelling plastic cannulae were
injected with the following macromolecular ma-
terials: 30% tapioca dextrin (TD), mol. wt.
2-10,000; 5% oxypolygelatin (OPG), mol. wt.
10-45,000; 3.5% polyvinylpyrrolidone (PVP),
mol. wt. 20-80,000. Tapioca dextrin, 150 ml/day
for 11 days, produced hydropic degeneration of the
epithelium of the proximal convoluted tubules.
No hyaline degeneration was detected in the
glomeruli or the blood vessels of the lungs. The
histological changes were reversible. OPG, 150
ml/day for 4.11 days, produced irreversible hy-
dropic degeneration of the convoluted tubules and
Henle’s loop, but the glomeruli were not involved.
Small discrete emboli were observed in the periph-
eral vessels of the lungs. An occasional giant cell
of the Langham’s type, heavily loaded with
lymphocytes, was observed in the liver. The phys-
ical deterioration of the animals was greater after
OPG than after TD. PVP, 75 ml/day for 9 days,
produced much more extensive damage than either

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

417

OPG or TD. The kidneys showed glomerular capil-
lary bed constriction, tubular occlusion and
‘sucrose-like nephrosis.? The hepatic cord cells
and liver sinusoids contained PVP deposits. PVP
deposits represented a 15% replacement of the
bone marrow bed and a 40% replacement of spleen
organ volume. There were also deposits in the
heart, lung, pancreas, adrenals and gastrointes-
tinal tract. There appears to be an inter-relation-
ship between the extent of and reversibility of tis-
sue deposition and damage and the molecular
weight range of the injected macromolecule. The
smaller sized TD caused less damage than either
OPG or PVP. Thus, it would appear that some
emphasis must be placed upon the ease of excre-
tion of a macromolecule by the kidney in any
evaluation of a plasma volume expander. However,
ability of the material to maintain the blood pres-
sure must also be considered. Perhaps a synthetic
molecule which can be metabolized as well as ex-
creted will be found; however, until such is avail-
able caution should be exercised in the adminis-
tration of macromolecular substances. (This
article is based on work performed under Contract
No. AT-04-1-GEN-12 between the Atomic Energy
Commission and the Univ. of California at Los
Angeles.)

1359. Effect of serotonin on cerebral oxygen
tension and evoked electrical activity in the
cerebral cortex. Victor DiSterano, DANIEL
E. Leary anp Isaac FELDMAN (introduced by
J. N. Stannarp). Div. of Pharmacology and
Toxicology, School of Medicine and Dentistry,
Univ. of Rochester, N. Y.

Studies have been carried out in the neurolog-
ically isolated suprasylvian cortex of the cat brain
to determine the effect of serotonin on induced
electrical activity. The evoked potentials from the
cortex obtained by electrical stimulation were de-
pressed by 5-10 ug of serotonin creatinine sulfate.
Depression was observed from 1 to 3 min. follow-
ing injection of the drug and persisted from 4 to 8
min. Little correlation was observed between
blood pressure and the depression of evoked cor-
tical potentials. Serotonin, a potent smooth muscle
stimulant in the periphery, might possibly exert
a local vasoconstrictor action in the cortex. Dim-
inution of blood flow and hypoxia could well ac-
count for the observed depression of evoked elec-
trical potentials. Consequently, measurements of
cortical oxygen tension in the right suprasylvian
cortex were carried out polarographically. Simul-
taneously, evoked electrical potentials from the
neurologically isolated left suprasylvian cortex
and the femoral blood pressure were recorded.
Ten to twenty ug of serotonin creatinine sulfate
invariably caused a fall in both the systemic blood
pressure and cortical oxygen tension. Depression

418 FEDERATION PROCEEDINGS

of the evoked cortical potentials, however, usually
did not occur until both the blood pressure and
cortical oxygen tension had returned to normal.
It is concluded that no correlation exists between
the depression of evoked cortical potentials and
oxygen tension lowering following the adminis-
tration of serotonin and that serotonin exerts a
real electrical effect on the cerebral cortex. (This
paper is based in part on work performed under
contract with the Atomic Energy Commission at
the Univ. of Rochester Atomic Energy Project,
Rochester, N. Y.)

1360. Effect of decreased barometric pressure
on radiation lethality in rats. JoHn DoULL.
U. 8. Air Force Radiation Lab. and Dept. of
Pharmacology, Univ. of Chicago, Chicago, Ill.

As part of a program designed to evaluate the
effects of various environmental factors on radia-
tion lethality in animals, adult male and female
Sprague Dawley rats were subjected to simulated
altitudes of 10,000, 20,000 and 30,000 ft. for various
periods before, during or following the adminis-
tration of lethal and sublethal doses of whole body
x-irradiation. The radiation factors were as fol-
lows: 250 KVP, 15 ma, target-skin distance 65 cm,
dose rate 40-43 r/min.). Neither pre- nor post-
irradiation exposure to a reduced barometric
pressure (12 hr/day at a simulated altitude of
10,000 or 20,000 ft.) for periods of 1, 7 or 14 days
significantly altered the survival time, weight loss
or mortality of rats given x-ray doses of 600, 800,
1000 and 1200 r. Increasing the duration of the
altitude exposure from 12 to 23 hr/day also failed
to alter radiation lethality in animals given these
doses of x-irradiation. Rats subjected to a simu-
lated altitude of 20,000 feet during the adminis-
tration of x-ray doses of 800, 1000 and 1200 r ex-
hibited a survival time, weight loss and mortality
similar to that occurring in animals given x-ray
doses of 600, 800 and 1000 r at ground level. One
hr. exposures to a simulated altitude of 30,000
feet either before or after these doses of x-ray did
not alter radiation lethality in rats but when the
animals were kept at this altitude during the x-ray
exposure the‘effective dose of radiation was re-
duced by approximately 300 r.

1361. Effect of Enheptin on endocrine system
and reproduction. Ropert H. DReEIsBacu.
Dept. of Pharmacology, Stanford Univ., San
Francisco, Calif.

Pino et al. (Proc. Soc. Exper. Biol. & Med. 37:
201, 1954) demonstrated depression of pituitary
gonadotropin production in chickens by the ad-
ministration of Enheptin (E, 40% 2-amino-5-
nitrothiazole) with associated inhibition of repro-
ductive functions. In our experiments, 12 female
mice were fed 1% E in the diet for 4 wk., being

Volume 16

caged with males for the last 3 wk. Five of the 12
had average litters, while 6 of 12 females given
regular food in the same way had litters. No effect
on the duration of estrus cycles was produced by
feeding 1% to mice or 0.1% to rats. Four rats fed
0.1% E were mated at the time of ovulation and
delivered average litters. Endocrine organ weight
(shown in table as average and range, mg/100 gm.
body weight) in 40-day-old rats after 2 wk. on diet
were determined after feeding.

| Enheptin 0.22% Thiouracil 0.1%
eee a |

Pituitary | 3.8 (2.5-5)| 4.1 (1-6) | 5 (4.5-8.5)
Ovary |\27 (24-32)|23 (15-42)}28 (20-41 )
Thyroid [10 (8-12) |19(17-21)}46 (30-66)
Adrenal (27 (21-35)|22 (19-26 )|22 (19-27)

Organ Control

The only significant effect on endocrine organ
weight was an increase in the weight of the thyroid
gland to about twice normal in E fed rats and to
four times normal in thouracil fed rats. Thyroids
in E and thiouracil fed rats showed histological
changes characteristic of antithyroid agents.
(Supported in part by the Planned Parenthood
Federation of America.)

1362. Action of cortisone on parenchymal
cells of mouse liver. CLARA E. Dunn,* ALLAN
D. Bass anp A. Hope McArpbie.* Pharmacology
Dept., Vandertilt Univ. School of Medicine,
Nashville, Tenn.

Previous investigations have provided evidence
that the desoxyribose nucleic acid (DNA) con-
centration of liver is reduced after 5 days of corti-
sone treatment. The processes involved in this re-
duction are not known. It has been suggested by
Bass, et al. (1952) that this decrease in DNA
concentration might be due to an alteration in the
ploidy distribution in the liver. Cortisone (1.0
mg/10-gm mouse/day) was administered to male
Swiss albino mice and the alterations of DNA,
RNA, nitrogen, glycogen, glucose, pyruvate, and
fat in the liver were followed at intervals for 2
wk. Binucleate cell counts and determinations of
DNA per individual nucleus were performed on
tissue samples from livers of cortisone treated
and pair fed control animals. Cortisone produces
an increase in DNA per average nucleus within
24 hr. which may be explained by a shift from the
normal ploidy distribution which is 25% diploid,
67% tetraploid and 8% octoploid; to a 9% diploid,
79% tetraploid, and 11% octoploid distribution.
These changes coincide with a sharp rise in intra-
cellular glycogen concentration and drop in RNA
and nitrogen concentration. Within 48 hr., there
is a rise above the normal percentage of diploid
nuclei observed and a corresponding drop in DNA

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March 1956

per average nucleus. Nuclei of higher ploidy classes
are seen More commonly in treated than in normal
mouse livers. Mechanisms involved in the altera-
tion in the distribution of ploidy classes in the
liver will be discussed.

1363. Hormonal effects on DDT storage in the
white rat. Wi_uiAM F. DurHAM, CIPRIANO
CuETO, JR. AND WAYLAND J. HayeEs, JR. (intro-
duced by Caru C. Pretrrer). Communicable
Disease Ctr., Public Health Service, Savannah,
Ga.

Sexual differences in the response of rats to DDT
dosage have been reported for chronic toxicity,
histopathology, and fat storage of the insecticide
and its derivative, DDE. The present study was
designed to investigate the physiological basis of
the two storage differences. Diethylstilbestrol
(DES) and testosterone propionate (TP) in peanut
oil solution were administered subcutaneously to
intact and gonadectomized rats fed 200 ppm DDT
in their diet and to control animals. As expected,
TP dosage increased the growth of female rats
while DES inhibited the growth of males. Either
hormone inhibited gonadal growth in young rats
of the opposite sex. Rats receiving either DDT or
hormone showed an increased liver weight/body
weight ratio as compared to control animals. This
effect did not appear to be due to the oil vehicle
in which the hormone was dissolved. DES in-
creased DDT and DDE storage in fat in the male
while TP decreased these values in the female.
Similar effects of hormone dosage were noted on
the ratio of DDE to total DDT-derived material
stored in the fat. Gonadectomy brought about, in
both sexes, changes in DDT and DDE storage
which were qualitatively the same as those result-
ing from hormone dosage, but lesser in magnitude.
There was some indication of an additive effect
between gonadectomy and hormone dosage on
DDT and DDE storage in the female rat but not in
the male.

1364. Resuscitation from ventricular fibrilla-
tion induced by chloroform and epineph-
rine. A. E. DyeR* anp J. K. W. Ferauson.
Dept. of Pharmacology, Univ. of Toronto, To-
ronto, Canada.

Some aspects of resuscitation from fibrillation
induced by the injection of epinephrine during ex-
posure to chloroform were studied in dogs. Using
a method previously described (DYER AND FERGU-
son, J. Pharmacol. & Exper. Therap. 112: 424, 1954)
in which electrical shocks were applied through the
intact thorax, resuscitation was successful in
96% of 144 cases of fibrillation. Following pre-
liminary experiments, only one loss occurred in
124 cases using shocks produced by a potential of
255 v. This voltage developed an average current

errr:

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

419

of 3.7 amp. (S.D. +0.7). Two shock intervals, 0.1
and 0.5 sec., appeared to be equally effective. On
several occasions similar shocks were adminis-
tered when the heart was exhibiting normal and
abnormal rhythms other than fibrillation. In no
cases were the effects injurious and in some im-
provement occurred. The observations made dur-
ing these experiments, together with the marked
stimulant effects of strong currents noted on res-
piratory movements, indicate that such currents
not only arrest fibrillation but may also assist re-
covery following defibrillation. Knowledge of the
current characteristics, which were found to be
within practical limits, permitted the construction
of a portable defibrillator which could be operated
from a normal line-potential of 110 v.

1365. Hypnotic effects of polyhydric alcohol-
chloral hemi-acetals. DonaLp K. Eckre.p,*
RicHarp TisLow AND JosEepH SEIFTER. Wyeth
Inst. for Med. Research, Radnor, Pa.

Chloral hemiacetals of some polyhydric alco-
hols such as glycols, glycerol, triethanolamine,
certain sugars, sugar-alcohols and polysaccharides
produced sedation and hypnosis as evidenced by
tolerance of side position in mice, rats or dogs.
These compounds were active by parenteral and
oral routes of administration respectively. The
dosage range of activity and toxicity in terms of
chloral content was similar. Of these compounds,
tetra-chloralpentaerythritol (CP) was studied in
more detail. This compound did not taste as bitter
and was not as irritating as chloral hydrate. The
hypnotic activity of CP and chloral hydrate were
alike with respect to hypnotic dose and duration
of sleep as determined in rats and dogs. In dogs as
well as in man CP had a smooth onset of action.
No undesirable side effects such as prostration or
hangover were observed. Nonhypnotic doses of CP
chloral hydrate produced sleep in mice and rats
when administered together with tranquilizing
agents of the chlorpromazine series.

1366. Effect of cell-free spleen extracts on
X-ray mortality of mice. FRrepRIcH ELLIN-
GER. Pharmacology Div., Naval Med. Research
Inst., Bethesda, Md.

A humoral factor, reducing x-ray mortality in
mice was demonstrated (Radiologia Clinica 23:
292, 1954) in saline extracts of quick frozen mouse
spleens, made cell free by centrifugation and bac-
terial filters. Spleens were removed from donor
animals 7 days after x-irradiation with Lp 30/14
days. That such humoral factor is also present in
extracts from normal mouse spleens is shown here.
The number of survivors in equal groups of mice
injected daily with 0.3 cc of saline or a variety of
spleen extracts for 5 days after exposure to the

420

LD 78/14 days is compared on the 20th day as
stated below:

Extract-treated Saline-treated

1 spleen/cc 22 of 72 15 of 72
2 spleens/cc 12 of 48 5 of 48
4 baby spleens/cc 5 of 24 3 of 24

Grand total 39 of 144 23 of 144

The difference between 39 and 23 is significant:
P < 0.05 (x? = 4.6246). No protection was found
using extracts from spleens removed 6 or 10 days
after exposure of the donor to tp 55/14 days, in
agreement with previous observations (16 survi-
vors out of 84 in both saline and spleen extract
groups). Differences in protein and NPN content
of unirradiated and irradiated spleen extracts
which seem to explain protection or failure to
protect will be shown.

1367. Content of adrenal medulla catechola-
mines as influenced by 3-hydroxytyramine.
Grorce L. Eximan (introduced by M. B.
CHENOWETH). Biochemical Research Dept., Dow
Chemical Co., Midland, Mich.

The quantitative separation of epinephrine,
nor-epinephrine, 3-hydroxytyramine, and related
compounds has been achieved using Dowex-50X8
(200-400 mesh) in the H form. The resin was
thoroughly washed with concentrated hydro-
chloric acid diluted 1:50. The solution of cate-
cholamines (pH 2-3) was introduced and then
eluted with this dilute HCl. Fractions were col-
lected and analyzed by either measuring UV ab-
sorption directly or by condensation with ethyl-
enediamine and measurement of the fluorescence
produced. (WEIL-MALHERBE AND Bone, Biochem.
J.51:311, 1952.) This method has been used in the
study of changes in the catecholamine content of
fresh beef adrenal medulla tissue. Such tissue was
incubated with 3-hydroxytyramine for 2-4 hr.
and then analyzed. The 3-hydroxytyramine disap-
peared and additional nor-epinephrine (represen-
tative data—control: 2.224. .04 mg/gm wet tissue;
test: 2.46+.94 mg/gm) was found. Of 3 experi-
ments, only 1 showed any increase in epinephrine
The increase of nor-epinephrine represented 53%
conversion of 3-hydroxytyramine to nor-epineph-
rine. The addition of a mixture of cytochrome c
(0.5 mm), ATP (9.2 mm), DPN (2.4 mM), pyruvate
(54 mm) and malate (7.5 mm) increased the cate-
cholamine content still further. Ascorbic acid did
not increase the catecholamine content over that
obtained with 3-hydroxytyramine, alone.

1368. In vitro effect of chlorpromazine on
human cholinesterases. Ervin G. Erpés,*
Francis F. Fotpes, Nora Baart* anp SYDNEY
P. SHanor.* Dept. of Anesthesiology, Mercy

FEDERATION PROCEEDINGS

Volume 16

Hosp., and Section on Anesthesiology, Dept. of

Surgery, Univ. of Pittsburgh, Pittsburgh, Pa.

In view of the widespread clinical use of chlor-
promazine, the knowledge of its effect on human
cholinesterases is of interest. It has been previ-
ously reported (CourvorsiER, S. et al. Arch.
internat. pharmacodyn. 92: 305, 1953) that chlor-
promazine in a concentration of 3 X 10 m inhibits
horse serum cholinesterase. In the present study
the inhibitory effect of chlorpromazine on the
hydrolysis of acetylcholine-Cl (ACh) and butyryl-
choline-Cl (BuCh) by human plasma and by con-
centrated human plasma cholinesterase (Cholase)
and the hydrolysis of ACh by human red cell
cholinesterase (RChE) was investigated by War-
burg’s manometric method and that of pro-
caine- HCl (proc.) by human plasma and Cholase
with Kalow’s u.v. spectrophotometric method (Ka-
Low, W. J. Pharmacol. & Exper. Therap. 104: 122,
1952) at pu 7.4 and 37°C. The substrate concentra-
tion in the plasma and Cholase experiments with
ACh and BuCh was 2.2 X 10-? and with procaine
5 X 10-° m. In the experiments with RChE the
ACh concentration was 3 X 10-° m. The Is values
found were: In human plasma with ACh substrate
3 X 10-° mM; with proc. substrate 4 X 10° m. In
Cholase with ACh substrate 2 X 10-°m; with BuCh
substrate 5 X 10-5 m; with proc. substrate 9 X
10-* m. For RChE with ACh substrate 3 x 10-¢
M. The Is values of chlorpromazine are of a
magnitude which may conceivably be obtained
in patients after its parenteral administration
in therapeutic doses. Jn vivo studies on the effect
of chlorpromazine on cholinesterases are now in
progress.

1369. Production of tremor and a Parkinson-
like syndrome by 1-4 dipyrrolidino-2-
butyne, “Tremorine.’ G. M. Everett, L. E.
Buiocxus,* I. M. SHeprperp* anv J. E. P. To-
MAN. Dept. of Pharmacology, Abbott Labs.,
North Chicago, and Dept. of Physiology, Chicago
Med. School, Chicago, Til.

In animals, sustained tremor is a rare phenome-
non of drug action (10 out of 10,000 drugs in our
experience). “Tremorine,’ when given in doses of
5-20 mg/kg by all routes, produces a marked sus-
tained tremor particularly of the head and limbs,
salivation, miosis and a slight muscular weakness
with rigidity in mice, rats, guinea pigs, dogs, cats
and monkeys. The picture is strikingly similar in
many features to human Parkinsonism particu-
larly in the monkey. Atropine and other anti-
Parkinson drugs are specific antagonists of “Tre-
morine,’ completely abolishing all signs of the
drug’s action, while hypnotics, anticonvulsants,
and ganglionic blocking agents have little effect
in doses below those producing severe depression.
Methantheline inhibits the peripheral cholinergic

che
pai

1e 16

it. of
Pa.

hlor-
man
revi-
Arch.
hlor-
ibits
tudy
. the
yryl-
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| cell
War-
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. To-
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ergi¢

March 1956

action but does not control the tremors, showing
the separation of the peripheral and central effects
of the drug. The site of action of the drug is prob-
ably subcortical. Temperature regulation is dis-
turbed and decerebrate animals still show tremor.

‘ Spinal animals show tremor above but not below

the level of section. The duration of ‘Tremorine’
action varies from 3 hr. in mice to 48 hr. in dogs
and monkeys. The latter two species usually die
of pulmonary complications if not treated with
anti-Parkinson drugs. The possible usefulness
of this drug in the study of the phenomena of
tremor and the screening of anti-Parkinson agents
opens these areas to further investigation.

1370. Bufotenine effects in humans. H. D.
Fapina,* E. L. Kropa,* J. R. HAwkins* AND
C. D. Leake. Pharmacology Lab., Ohio State
Univ. and Battelle Memorial Inst., Columbus.
Bufotenine (N,N -dimethyl-5-hydroxytrypta-

mine) with creatinine sulfate in physiological sa-

line solution was injected intravenously into hu-
man volunteers, in amounts varying from 0.06 to

to 0.25 mg/kg and over times ranging from 3 to 20

min. With the higher dosages and more rapid in-

jections there was reported prompt sensations of
tingling spreading from the head and neck to the
extremities, with constriction of the chest, visual
disturbances, color hallucinations, nausea and
mental confusion. We noted purpling of the skin
in the head and neck, retching, vomiting, nystag-
mus and mild disorientation. Respiration, pulse,
blood pressure and knee jerk varied slightly with

a tendency to increase. Within a few minutes after

stopping the injection there was gradual relaxa-

tion, lassitude and withdrawal, followed by a slow

return to usual mental awareness, and then by a

significant euphoria lasting for several hours.

There was some reduction of blood oxygen content

during the initial symptoms. With the lower dos-

ages and slower administration, the initial symp-
toms were absent, but the euphoric state de-
veloped directly.

1371. Effect of acidosis and alkalosis on the
reduction of renal cellular protein-bound
sulfhydryl concentration produced by a
mercurial diuretic. A. Farau, R. Kruse,* C.
H. BenpER* anp E, Carruny.* Dept. of Phar-
macology, State Univ. of New York, Upstate Med.
Ctr., Syracuse, N. Y.

Previous studies on rat and dog kidneys with a
cytophotometric technique have shown that the
mercurial diuretic, mersalyl, produced a reduction
in protein-bound sulfhydryl (PBSH) concentra-
tion of cells in the terminal part of the proximal
tubules in Henle’s loop and collecting ducts. No
changes were found in the cells of the convoluted
parts of the proximal tubules or the distal con-

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

421

voluted tubules. Our studies have now been ex-
tended to 4 other organic mercurial diuretics and
in all instances the changes observed were qualita-
tively and quantitatively similar to those seen
with mersalyl. Mercury bichloride was quantita-
tively more effective than the organic mercury
compounds although qualitatively the changes
were the same. It can be shown that during acido-
sis the mersalyl induced reduction in PBSH of the
cells of the terminal part of the proximal tubule
was 2-3 times as great as that seen in alkalotic rats
or dogs. In the cells of the collecting ducts bicar-
bonate administration produced a marked reduc-
tion in PBSH concentration and mersalyl pro-
duced a partial reversal of this reduction in PBSH.
During the administration of acid, PBSH concen-
tration was normal and mersaly] produced a reduc-
tion in the PBSH concentration of cells of the col-
lecting tubules.

1372. Acetylcholine effects on the nictitating
membrane. GLoRIA C. FEENEY,* MARGARET
AveRY* AND THEODORE Koppanyi. Dept. of
Pharmacology, Georgetown Med. School, Wash-
ington, D.C.

With acetylcholine we produced contractions of
the nictitating membrane even after complete
ganglionic blockade by hexamethonium. Morison
and Acheson (Am. J. Physiol. 12: 149, 1938) re-
ported that acetylcholine produced contractions
of the membrane after superior cervical ganglion-
ectomy. These 2 observations suggested a series
of experiments designed to separate the musca-
rinic and nicotinic components of the acetylcho-
line-induced contractions of the nictitating mem-
brane. Cats (chloralose anesthesia) were used. The
nictitating membrane contractions were recorded
isotonically. Acetylcholine was injected intrave-
nously in doses of 4-500 wg/kg. In 2 series of experi-
ments, there were 2 control runs to determine the
dose-effect relationship. a) Cats received intra-
venously from 0.2 to 1 mg/kg atropine sulfate.
These doses abolished nictitating membrane con-
tractions by acetylcholine in the majority of cases.
Even maximal contractions were abolished. After
the atropine experiments, 5 mg/kg of hexametho-
nium was given intravenously. In a few animals,
only, did hexamethonium reduce the height of
contractions. 6) Hexamethonium ion (5 mg/kg)
was given first in another series of cats. This dose
in the majority of the animals did not reduce the
height of the contractions even after large doses
of acetylcholine. In very few animals was hexa-
methonium effective. Subsequent atropinization
in the majority of the animals completely abol-
ished acetylcholine contractions unaffected by
hexamethonium. In a few animals, atropine abol-
ished the contractions following small doses of
acetylcholine, but not after larger doses. Residual

422

contraction of the membrane still present after
light atropinization represents the nicotinic com-
ponent and residual contractions after hexameth-
onium administration, the muscarinic component.
The relative ineffectiveness of hexamethonium to
abolish or diminish the contractions suggests that
acetylcholine in the majority of cats produces its
effects by muscarinic action on the cholinergic
sympathetic endings in the muscle of the nictitat-
ing membrane. The existence of such fibers was
already discussed by Bacq and Fredericq (Compt.
Rend. Soc. Biol. 117: 482, 1934).

1373. Chronic toxicities of two food colors,
FD&C Red No. 32 and FD&C Orange No. 2.
O. Gartu FirzHucH, ArTHUR A. NELSON AND
ANNE R. Bourke.* Div. of Pharmacology, Food
and Drug Admin., Dept. of Health, Education
and Welfare, Washington, D. C.

In a 2-year chronic experiment weanling rats
were started on dosage levels of 0.25 and 0.1%
FD&C Red No. 32 in their diet. The feeding of
0.25% Red No. 32 caused early deaths, changes due
to inanition, moderate to marked subacute liver
damage and enlargement of the common bile duct.
The dosage level of 0.1% caused an increase in
mortality, decreased growth rate, slight to mod-
erate liver damage and pronounced right-sided
heart damage. Sixteen dogs were given Red No. 32
at dosage levels ranging from 0.01 to 0.2% for pe-
riods of 26 days-2 yr. Emaciation of varying degree
was consistent at the higher levels. Microscopi-
cally, atrophy of internal organs paralleled ema-
ciation. The most frequently affected organs were
liver, spleen, lymph nodes, bone marrow, genital
organs and skeletal muscle. In addition, effects
attributed to treatment included slight fatty
change in the liver, dystrophic changes in skeletal
muscle and some hemosiderosis in liver and bone
marrow. A less extensive study with a closely re-
lated color, FD&C Orange No. 2, showed changes
in rats and dogs similar to those produced by like
concentrations of FD&C Red No. 32.

1374. Interrelationship of suxamethonium,
suxethonium and succinylmonocholine and
human cholinesterases. FRANcis F. Foupss,
GERTRUDE R. van HeEEs,* SypNey P. SHANOR*
AND Nora Baart.* Dept. of Anesthesiology,
Mercy Hosp., and Section on Anesthesiology, Dept.
of Surgery, Univ. of Pittsburgh, Pittsburgh, Pa.
The enzymatic hydrolysis of suxamethonium

(SDCh), suxethonium (SEDCh) and succinyl-

monocholine (SMCh) by a human plasma eholines-

terase concentrate (Cholase) and human red cell
cholinesterase (RChE) as well as the inhibitory
effect of these compounds on the enzymatic hy-
drolysis of acetylcholine (ACh) by the same en-
zymes was investigated with Warburg’s mano-
metric method at pH 7.4 and 37°C. In substrate

FEDERATION PROCEEDINGS

Volume 16

concentrations ranging from 1.6 X 10-?m to3.0 X
10~* m neither SDCh, SEDCh nor SMCh were hy-
drolyzed by RChE. The maximum observed veloc-
ities (Vm) forSDCh, SEDCh and SMCh with sub-
strate concentrations of 2.2 x 10-2 M, in Cholase,
were, respectively, 2.1 X 10-2, 3.5 x 10-* and 6.0 X
10-° m/l. X min. This compared with a Vm value
of 7.9 X 107! m/l. X min. for ACh. The Ky values
of SDCh, SEDCh, SMCh and ACh in Cholase were
1.8 X 10°, 3.1 X 10-4, 2.9 X 10°? and 1.8 X 10%
M/l., respectively. The Is values for SDCh,
SEDCh and SMCh for the hydrolysis of ACh by
Cholase were, respectively, 6.3 X 10-4, 4.2 K 1074
and 1.4 X 107 m/l. The same values for RChE
were 8.3 X 1071, 2.5 X 10-4 and 2.3 X 10°? m/l.

1375. Absorption, distribution and fate of
sulfaethylthiadiazole administered orally
and intravenously. E. L. Foutz, J. V. Swin-
TOsKy* AND M. J. Rosrnson.* Section of Infec-
tious Diseases, Dept. of Medicine and the William
Pepper Lab. of Clinical Medicine, Univ. of Penn-
syluania School of Medicine, and Smith, Kline
and French Labs., Philadelphia, Pa.
Sulfaethylthiadiazole is rapidly absorbed within

the first 2 hr. after oral ingestion. In adult subjects
given a 4.0 gm oral dose, peak blood concentra-
tions of 14-20 mg % were noted in 2 hr.; smaller
doses of 2.0 gm produced levels in excess of those
required for bacteriostasis. Intravenous doses of
2.0 gm generally gave blood concentrations in ex-
cess of 10 mg %. The half-life for disappearance
from the blood stream by renal excretion, as deter-
mined by blood concentrations, was approxi-
mately 7-8 hr. Maintenance doses of 1.0 gm at
either 4 or 6-hr. intervals were effective in main-
taining therapeutic blood levels. Calculations sug-
gested that after equilibrium had occurred, aver-
age body distribution volumes of 17-20% were
occupied by the drug. Acetylation of the drug
occurred; however, urinary recoveries generally
demonstrated less than 10% acetylation. Elimina-
tion by urinary excretion occurred at approxi-
mately a first order rate and was essentially
complete (90-100%) 72 hr. after a single dose, with
approximately 75-80% recovery in the first 24 hr.
Nocrystalluria, hematuria, or renal complications
were encountered in these trials, which were con-
ducted without benefit of alkali therapy and with
only moderate fluid intakes. Sulfaethylthiadiazole
appears to be an effective and safe antimicrobial
agent.

1376. Acceleration of reduction of foreign
compounds in vitro and in vivo by various
dyes. James R. Fouts* anp BERNARD B. Bro-
pIE. Lab. of Chemical Pharmacology, Natl. Heart
Inst., Bethesda, Md.

A number of compounds have been shown to
prolong the action of a variety of drugs by block-

Odet® &> @s8non

me 16

3.0 %
re hy-
veloc-
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6.0 X
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values
e were
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Ch by
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Swin-
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imina-
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ations
e con-
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. Bro-

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March 1956

ing their metabolic transformation. However, sub-
stances that increase the rate of biotransformation
of drugs have received little attention. Such sub-
stances would have potential value in the treat-
ment of toxicity of foreign compounds. The pres-
ent studies show that the enzymatic reduction of
aromatic nitro compounds to the corresponding
amines is markedly accelerated by a number of re-
versibly oxidizable compounds including Janus
Green B, Indigo, riboflavin, flavin adenine dinu-
cleotide and riboflavin-5-Phosphate (FMN). For
example, 10-? m FMN increases the rate of reduc-
tion of p-nitrobenzoic acid in mouse and rabbit
liver homogenates about 300%. FMN is also effec-
tive in vivo; the rate of reduction of p-nitrobenzoic
acid is markedly increased in mice pretreated with
large amounts of FMN. Possible mechanisms of
this acceleration will be discussed. In addition,
data on the effects of FMN on the reduction of a
number of drugs in vitro and in vivo will be pre-
sented.

1377. Minimum dose of barbiturates required
to produce physical dependence. H. F. Fra-
sER, Harris IsBELL, A. WikuER, R. E. BELLE-
VILLE,* C. F. Essia* anp H. E. Hitu.* Nail.
Inst. of Mental Health, Addiction Research Ctr.,
PHS Hosp., Lexington, Ky.

Twenty-three partially tolerant patients re-
ceived either 0.8 or 0.6 gm of secobarbital daily for
42-120 days. When the drug was withdrawn ab-
ruptly 1 of 5 receiving 0.8 gm and 2 of 18 receiv-
ing 0.6 gm daily had grand mal convulsions; none
developed delirium. In another experiment, 10
nontolerant subjects received 0.4 gm secobarbital
and 8 nontolerant patients received 0.4 gm pento-
barbital daily. All were observed for 30 days prior
to, 90 days during and 45 days after, drug adminis-
tration. Results with 0.4 gm secobarbital and with
0.4 gm pentobarbital were similar. Initially, 16 of
these 18 subjects showed marked, and 2 showed
mild intoxication (incoordination, dysarthria,
confusion, poor judgment and various mood
changes). These changes subsided rapidly from
the 4th through the 7th day of drug administra-
tion. After 80 days of drug intake there was no sig-
nificant impairment of function as judged by per-
formance tests (coordination, reaction-time and
pursuit rotor). After abrupt withdrawal of bar-
biturates none of these 18 subjects given 0.4 gm
developed convulsions or delirium; they showed
only transient tremor and/or mild anxiety with
insomnia. One showed frequent bursts of high
voltage slow waves in EEG. Following withdrawal
of 0.2 gm secobarbital from 2 subjects who had re-
ceived it nightly for a year, no significant symp-
toms were observed. Conclusion: in healthy males,
a chronic dose of greater than 0.4 gm secobarbital
or pentobarbital daily is required to produce a

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

423

clinically significant degree of physical depend-
ence.

1378. Addictive potentialities of hexamethyl-
eneimines. H. F. Fraser. Natl. Inst. of Mental
Health, Addiction Research Ctr., PHS Hosp.,
Lexington, Ky.

Neither objective signs nor subjective symp-
toms of morphine-like effect were observed in non-
tolerant former addicts after administration of the
following compounds: 1-methyl-4-phenyl-4-car-
bethoxy hexamethyleneimine (I) in doses of 25-
150 mg s.c. or 50-300 mg orally; 1,3-Dimethyl-4-
phenyl-4-carbethoxy hexamethyleneimine (II) in
doses of 5-200 mg s.c.; and 1,2-Dimethy]-4-phenyl-
4-carbmethoxy-hexamethyleneimine (III) in doses
of 5-80 mg s.c. or 40-100 mg orally. Di-alpha-1,3-
Dimethy] -4-pheny]-4-propionoxy-hexamethylene-
imine (IV) in doses of 75-150 mg induced objective
signs and subjective svmntoms of morphine-like
effect roughly equivalent to those which follow
15-30 mg of morphine but of shorter duration. I, IT
and III in doses of 100, 150 and 150 mg s.c. every
4-6 hr. were ineffective in suppressing symptoms
of abstinence in patients who were addicted to
240-280 mg of morphine daily. All addicted pa-
tients who received multiple doses of I, II and III
developed nervousness, insomnia, twitches and
tremors which persisted for 12-24 hr. after the
drugs were discontinued. One addicted patient
developed signs of vascular collapse and pulmo-
nary edema after receiving 150 mg of I. Similar but
milder episodes occurred in 2 other patients. Two
hundred mg of IV every 3 hr. suppressed absti-
nence completely and no toxic symptoms were ob-
served. Addictive potentialities of I, II and III ar?
either low or nonexistent; complete evaluation,
however, was impossible because of the central
nervous system excitation and other toxic effects
induced by these drugs. IV, on the other hand, has
addiction liability at least equal to that of meperi-
dine and approaching that of morphine.

1379. Effect of auxins and antiauxins on the
growth of fungi. [An M. FRASER AND B. Fusi-
KAWA (introduced by CHartes M. GRUBER).
Dept. of Pharmacology and Exptl. Therapeutics,
College of Med. Evangelists, Loma Linda, Calif.
In an attempt to elucidate possible growth regu-

lating mechanisms which might be involved in the

chemotherapy of fungus infections, the effect of
the higher plant auxins and antiauxins on the
growth of several fungi has been investigated. The
growth of Saccharomyces cereviseae in a synthetic
medium is inhibited by both auxins and antiauxins
at concentrations between 10~‘ m and 107? y, al-
though the antiauxins tend to be more active.

The concentration required to produce 50% in-

hibition is influenced by the px of the medium.

Interaction of several auxins and antiauxins at a

424

range of concentrations has been studied without
observing evidence of the reversal of antiauxin
inhibition by auxins. The growth of a strain of
Psalliota bispora on a synthetic medium is mark-
edly enhanced by certain concentrations of the
auxin indole-3-acetic acid but is not significantly
promoted by the auxins naphthalene acetic acid
and 2,4-dichlorophenoxyacetic acid. The possible
role of indole-3-acetic acid in the growth of fungi
will be discussed.

1380. Myelin degeneration in chickens with
subacute administration of organic phos-
phorous insecticides. JoHN P. FRAWLEY,
Rosert E. Zwickey* anp Henry N. Fuyar.*
Div. of Pharmacology, Food and Drug Admin.,
Washington, D. C.

Myelin degeneration was first associated with an
organic phosphorous compound by Smith et al.
(Public Health Rep. 45: 1703, 1930) who demon-
strated that the ‘Jakeleg paralysis’ syndrome of
that time was due to tri-ortho-cresyl phosphate
(TOCP). More recently Bidstrup et al. (Brit. Med.
J. 1: 1068, 1953) reported similar cases from an
organic phosphorous insecticide manufactured in
Britain, mipafox. Since both compounds are 7n vivo
cholinesterase inhibitors, it was reasoned that per-
haps other anticholinesterase insecticides used in
this country might produce this effect with sub-
acute administration. The chicken was chosen for
these studies since it most closely resembles man
in response to this effect. The insecticides were
mixed in commercial laying mash and fed to year
old chickens. Dosage was started considerably
below the acute lethal dose and increased at sev-
eral week intervals until either death or paralysis
occurred. After increasing the feeding level of
parathion and systox from 100 ppm to 1600 ppm
over an 18-wk. period death occurred in all birds
without any indications of paralysis or micro-
scopic nerve damage. Feeding malathion for 15 wk.
at levels up to 10,000 ppm was lethal to all birds,
but only one showed muscle weakness. However,
none of these animals showed nerve damage on
histopathological examination. Feeding of EPN
up to 600 Spm and TOCP at 2500 ppm gave rise to
definite clinical signs of paralysis in all animals
and histopathology revealed myelin degeneration
in all cases.

1381. Chemical measurement of amethopterin
in plasma and urine. M. V. FREEMAN* AND C.
G. Zusrop. Natl. Cancer Inst., Bethesda, Md.
The method has been developed from the ob-

servations of Allfrey, et al. (J. Biol. Chem. 178: 465,

1949) that folic acid upon oxidation yields a highly

fluorescent compound. The concentration of ame-

thopterin in an aqueous solution bears a linear
relationship to the fluorescence of its oxidized

FEDERATION PROCEEDINGS

Volume 16

product. The sensitivity is such that .01 mug/ml
can be determined with accuracy. Fasting plasma
and urine blanks show little or no increase in
fluorescence upon oxidation. The method measures
a material whose fluorescence is activated at ap-
proximately 360, which fluoresces at approxi-
mately 460 and which will increase in fluorescence
in this range upon oxidation. The recovery of
amounts added to plasma and urine is 100%+10%
over a wide range of concentrations. Extension of
these studies to man shows that the amethopterin
is readily absorbed from the gastrointestinal tract,
that a large proportion is excreted rapidly into the
urine Plasma concentrations are maintained only
2 hr. after a 2.5 mg dose and 4-6 hr. after a 5-10 mg
dose. The volume of distribution indicates that
amethopterin is limited to the extracellular fluid,

1382. Convenient procedure for synthesis of 1-
C' amino acids. HERBERT J. Fromm (intro-
duced by B. DeBoer). Dept. of Biochemistry,
Univ. of North Dakota Med. School, Grand Forks,
N. D.

The synthesis of labeled amino acids generally
necessitates the use of specific procedures for each
amino acid. A standard 5-step method for the
synthesis of 1-C!* amino acids has been investi-
gated. Phthaliminoacetic acid was prepared by
fusing glycine and phthalic anhydride. Silver
phthaliminoacetate was obtained by treating the
acid with aqueous AgNO; at pu 7.0. A small
amount of N-bromomethylphthalimide along with
the parent acid was obtained by treating the dry
silver salt in anhydrous CCl with bromine. The
mixture, reacted with C'*-labeled NaCN, yielded
the labeled nitrile. 1-C'* glycine was obtained by
refluxing the product with acid. Success in pre-
liminary studies has been achieved using this pro-
cedure for the synthesis of 1-C'*-labeled alanine
and methionine. Carboxy]-labeled fatty acids were
prepared by simply subjecting the silver salts of
the acids to the synthesis described for amino
acids.

1383. Cardiodecelerator and antiaccelerator
effects of sparteine on the dog heart-lung
preparation. J. FuEeNTES (introduced by O.
Krayer). Dept. of Pharmacology, Harvard Med.
School, Boston, Mass.

It is well known that sparteine exhibits negative
chronotropic and negative inotropic action in the
vertebrate heart (Heffter Handbuch der Experi-
mentellen Pharmakologie, Volume II, 2, p. 724,
1924). The negative chronotropic effect was rein-
vestigated in the heart-lung preparation of the
dog (HLP) with a blood volume of about 1 liter.
Doses of 20-50 mg of sparteine decrease the rate/
min. by 15-40 beats. Larger doses cause a strong
negative inotropic effect and occasionally produce

res
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an

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138

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myug/ml
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ease in
easures
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escence
very of
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ision of
opterin
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nto the
ed only
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ir fluid.

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(intro-
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ng with
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March 1956

disturbance of rhythm. Ouabain (20-50 yg) pre-
vents the negative inotropic effect thereby making
possible the administration of doses up to 100 mg
of sparteine and a correspondingly larger decrease
in rate/min. (45-69 beats). The heart rate increase
caused in the HLP by the continuous infusion of
3ug of epinephrine/min. or by a total dose of 3 mg
of ephedrine, can be completely antagonized by
sparteine in the presence, as well as in the absence,
of atropine. In 7 experiments under ephedrine,
the dose of sparteine causing 50% inhibition of ac-
celeration was 40 mg (range 28-60 mg). In this
regard the pharmacological action of sparteine
resembles that of veratramine (KRAYER AND Ov-
nisson, J. Pharmacol. & Exper. Therap. 112: 341,
1954). Judging from the ephedrine experiments,
and on a molar basis, the antiaccelerator potency
of sparteine is approximately 1/250 of that of
veratramine.

1384. Isolation of a purified form of bound
morphine from human urine. James M.
Fustmoro (introduced by E. Lrone Way). Dept.
of Pharmacology and Exptl. Therapeutics, Univ.
of California Med. Ctr., San Francisco.

Bound morphine in a highly purified state has
been isolated from the urine of addicts given mor-
phine. Preliminary separation of bound morphine
was accomplished as previously described by satu-
rating the urine K,CO;. The resulting red gum,
which precipitates, contains the major fraction of
the bound morphine present in the urine. Solution
of the gum in methanol, then water and treatment
with charcoal and subsequent elution of the char-
coal with acetic acid results in considerable re-
moval of extraneous material from the bound
morphine (Fustmoto AND Way, Federation Proc.
13: 356, 1954). More recently further purification
of the acetic acid eluate has been effected by ap-
plying continuous flow paper electrophoresis fol-
lowed by countercurrent distribution. The sepa-
rated fractions thus obtained, which contained
high concentrations of bound morphine, were
pooled and treated with dinitrofluorobenzene to
remove contaminants which react with ninhydrin.
After removal of the excess dinitrofluorobenzene,
an aqueous solution of the bound morphine was
streaked across Whatmann #1 filter paper and
chromatogrammed with acetic acid/n-butanol/
water (30/100/45). Subsequent elution with water
and evaporation of the eluate yielded 44 mg of an
isotropic crystal-like material. Studies to date on
this material indicate that it is different from
morphine monoglucuronide which has been iso-
lated from dogs (Woops, J. Pharmacol. & Exper.
Therap. 112: 158, 1954). (Supported in part by the
Natl. Insts. of Health and the Med. Research
Committee, Univ. of California Med. Ctr., and in

ear >

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

425

cooperation with authorities of the Addiction Re-
search Ctr., Lexington, Ky.)

1385. Depression of contractile force of iso-
lated auricles by ryanodine. Roserr F.
Furcueort AND TaIsiJA DE GUBAREFF.* Phar-
macology Dept., Washington Univ. School of
Medicine, St. Louis, Mo.

Exposure of isolated electrically driven left au-
ricles (guinea pig, rat, rabbit or monkey) to about
2 X 10-* ryanodine for about 20 min. significantly
reduces contractile force. Higher concentrations
can reduce force to practically zero. After washout
of ryanodine, its effect is essentially irreversible
for several hours. The decrease in force is not ac-
companied by any significant fall in high-energy
phosphates. Although the recovery phase of the
action potential is prolonged by ryanodine, it ap-
pears unlikely that this change is responsible for
the decrease in force. The action of ryanodine was
investigated in relation to the following scheme
(presented at Pharmacology Society meeting,
Sept. 1955) for mammalian cardiac rouscle (CM):

1
Unactivated relaxed CM cee? Activated relaxed CM
(4

)

Reactions (2) and (3) represent contraction and
relaxation processes of a single beat. Contractile
force depends largely on extent of activation of
CM at the time the action potential (AP) sets off
contraction. The rate of activation between beats
(reaction (1)) may be greatly accelerated by in-
creases in frequency, or drugs such as epinephrine,
strophanthin and calcium. Ordinarily activation
by reaction (1) is not appreciably counteracted
by deactivation due to reaction (4). However,
ryanodine appears to greatly accelerate reaction
(4), thus reducing contractile force. Evidence for
this hypothesis will be presented. (Supported by
grant from St. Louis Heart Assoc.)

1386. Metabolic products of C'‘-labeled atro-
pine in mice. JoHN D. GABOUREL AND ROBERT
E. Gosse.in (introduced by E. A. MayNnarp).
Dept. of Pharmacology and Toxicology, Univ. of
Rochester School of Medicine and Dentistry,
Rochester, N. Y.

After i.v. injection of atropine, labeled with C'*
in the alpha position of the tropic acid moiety,
radiochromatography showed at least 7 metabolic
products in mouse urine (Rr = 0.14, 0.22, 0.36,
0.57, 0.68, 0.72 and 0.82). An 8th component (Rr =
0.93), tentatively identified as tropic acid, was
found in urine specimens formed from 6 to 8 hr.
after injection; it constituted approximately 1%
of the injected dose. Of the 8 components sepa-
rated, only product Rr 0.72 and product Rr 0.82

426

possessed mydriatic activity in the mouse eye.
The latter has been identified as atropine (J. Phar-
macol. & Exper. Therap. 115: 217, 1955). Four of
these 8 products accounted for most (85-90%) of
the urinary C'* (Rr = 0.14, 0.22, 0.72 and 0.82). In
consecutive urine specimens, product Rr = 0.82
(atropine) was the largest component during the
Ist half hour; product Rr = 0.22 was the greatest
during the 2nd hour; and product Rr = 0.14 was
the largest during the 3rd hour and thereafter (up
to at least 10 hr.). It is inferred from these data
that product Rr = 0.22 is a precursor of Rr =
0.14. In urine specimens obtained after the 3rd
hour, relative concentrations of the various com-
ponents remained constant at the following levels:
Rr 0.14 = 34%, Rr 0.22 = 32%, Rr 0.72 = 10%,
and Rr 0.82 = 13%. Preliminary experiments show
no appreciable difference in atropine metabolism
in the DFP poisoned mouse (5 mg/kg).

1387. Constrictor and dilator cells in sympa-
thetic ganglia. Ropert W. GarpirerR,* BENE-
pict E. Apreu, Witi1am M. ALEXANDER* AND
Auice B. Ricuarps.* Research Dept., Pitman-
Moore Co., Div. of Allied Labs., Indianapolis,
Ind.

A series of acute experiments on anesthetized
(pentobarbital) dogs gave evidence supporting
Shaw’s contention (Australian J. Exper. Biol. &
M. Sc. 26: 139, 1948; ibid., 27: 289, 1949) for 2 phar-
macologically distinct cell types in sympathetic
ganglia. In atropinized animals with adrenals re-
moved, intravenous doses (1-2 mg/kg) of acetyl-
choline that gave pressor effects prior to adrenal-
ectomy, caused depressor, biphasic or slight
pressor responses; the direction or predominate
direction was inversely related to the blood pres-
sure level at the time of injection. In atropinized
intact dogs, hexamethonium (2.5 mg/kg) some-
times transiently reversed the pressor response to
acetylcholine. However, following adrenalectomy
and independent of the blood pressure, this re-
sponse was consistently reversed by hexametho-
nium. Conversely, the reduced pressor effect of
nicotine, following adrenalectomy was still further
depressed but never reversed by hexamethonium.
These data indicate that the pressor effect from
acetylcholine is almost entirely due to adrenal
medullary stimulation while that to nicotine is
due in greater part (60%) to ganglionic stimula-
tion. Moreover, the principal ganglionic action of
acetylcholine, although dependent on the existing
blood pressurse, is to stimulate dilator cells. The
acetylcholine reversal following hexamethonium
suggests that the latter is ineffective in blocking
the dilator cells but effective in giving a short-
lived blockade at the adrenal. Furthermore, the
nicotine responses after hexamethonium denote

FEDERATION PROCEEDINGS

Volume 16

that the constrictor cells in sympathetic ganglia
are sites of action common to both compounds,

1388. Prolongation and enhancement of the
tranquillizing action of reserpine by glu-
cose in the monkey. R. D. GaucHat* ANp R,
M. FeatuHerstone. Depts. of Pediatrics and
Pharmacology, College of Medicine, State Univ.
of Iowa, Iowa City.

Twelve Cynomolgus monkeys were housed in
individual cages suspended by springs in a manner
which permitted measurement of: 1) intake of
liquid feedings, 2) urinary output and 3) spon-
taneous activity by kymographic recording of ver-
tical fluctuatings of the cage-box. Observers with-
out knowledge of experimental schedules recorded
estimates of each animal’s level of activity at regu-
lar times daily. According to a randomized pro-
gram designed to test no more than 6 animals per
day, each animal was subjected to 16 tests over an
8-wk. period. On 5 of the test days a standard dose
of reserpine (Serpasil, CIBA) was given intra-
muscularly. The other 11 test situations exposed
the animal to reserpine variously combined with
compounds known to be significant in carbohy-
drate metabolism, or to control tests. A standard
pattern of response to reserpine was determined,
characterized by approximately 50% reduction in
fluid intake and output and marked reduction in
both observed and kymographically recorded ac-
tivity on the test day, with resumption of normal
intake, output and activity within 24 hr. Results
of the other tests, using reserpine plus carbohy-
drate factors, indicated that intravenous glucose
definitely enhanced and prolonged the ‘tranquil-
lizing’ action of reserpine when given with reser-
pine simultaneously. However, glucose 1 or 4 hr.
after reserpine neither enhanced nor prolonged the
effect of reserpine. Intravenous sucrose similarly
augmented the activity of reserpine. Insulin did
not modify reserpine’s action, but seemed to re-
duce the effect of glucose when all 3 were given at
the same time.

1389. Metabolic activity of 3,3’-diiodothyro-
nine and 3,3’ - diiodo - 5 - bromothyronine,
CuatMers L. GemmiLu. Dept. of Pharmacology,
Univ. of Virginia, Charlottesville.

Following the complete synthesis of 3-iodo-
thyronine and 3-iodo-5-bromothyronine, these
compounds were iodinated by 1 step iodination to
3,3’-diiodothyronine and 3,3’-diiodo-5-bromo-
thyronine. Details of the syntheses and proofs of
structure have been submitted by Gemmill, An-
derson and Burger to the J. Amer. Chem. Soc. for

a X
Lon
the
vess
tung
chre
fluol
back
Ino
of e:
com!
pher
were
mu,
of p
fluor
taine
(mol
and

ratio
tibof
mixe
was {
riboft
tive

phen:
was

ANP
of p-

publication. Both of these latter compounds we
tested for biological activity by oxygen consump-
tion of thyroidectomized rats and by the anti-

tions
of th
fluore

goiter test. It was found in the antigoiter test thatfof AN

ume 18

vanglia
ounds.

of the
y glu-
AND R,
cs and
> Univ.

ised in
manner
sake of
) spon-
of ver-
‘s with-
corded
it regu-
ed pro-
1als per
over an
rd dose
| intra-
»xposed
ad with
irbohy-
sandard
rmined,
etion in
‘tion in
ded ac-
normal
Results
urbohy-
glucose
-anquil-
h reser-
or 4 hr.
ged the
milarly
ulin did
1 to re-
riven at

»thyro-
ronine,
acology,

3-iodo-
, these
ation to
-bromo-
roofs of
ill, An-

Soc. for
ids we

ynsump-
he anti
est thal

March 1956

(+) 3,3’-diiodothyronine had approximately 4%
of the activity of (—) 3,3’,5-triiodothyronine. In
contrast, (+) 3,3’-diiodo-5-bromothyronine had
approximately 45% of the activity of the (—)
3,3’,5-triiodothyronine. In the studies on oxygen
consumption of thyroidectomized rats, (+) 3,3’-
diiodothyronine had little activity while (+) 3,3’-
diiodo-5-bromothyronine had approximately 40%
of the activity of (—) 3,3’,5-triiodothyronine.
These results demonstrate that iodine in position
5 of the thyroxine molecule is important for bio-
logical activity and that bromine may be substi-
tuted for iodine in position 5 with retention of
metabolic activity.

1390. Effects of thyroxine and riboflavin on
the transfer of energy. CHALMERS L. GEM-
MILL. Dept. of Pharmacology, School of Medicine,
Univ. of Virginia, Charlottesville.

In order to study the effects of the transfer of
excitation energy to thyroxine, riboflavin and
other molecules of biological interest, the Beck-
man DK-1 spectrophotometer was modified in
order to record automatically the fluorescent spec-
tra (GEMMILL, Anal. Chem. In press). Light from
a Xenon are was passed through a Bausch and
Lomb monochromator and then through a hole in
the back plate. The solution was placed in a quartz
vessel in the position formerly occupied by the
tungsten light. The entering light from the mono-
chromator caused the solution to fluoresce. The
fluorescent light is picked up by the mirror on the
back plate and passed into the spectrophotometer.
In order to determine the efficiency of the transfer
of excitation energy between compounds, various
combinations of phosphors were used. When p-ter-
phenyl and alphanaphthylphenyloxazole (ANPO)
were used in toluene with the exciting light at 280
mu, there was marked reduction of the fluorescence
of p-terphenyl and marked augmentation of the
fluorescence of ANPO. Similar results were ob-
tained with p-terphenyl and diphenylhexatriene
(molar ratio of 1136) and with 2,5-diphenyloxazole
and 1,4-di-2(5-phenyloxazolyl)benzene (molar
ratio of 9737). Various amounts of riboflavin and
tiboflavin-5-phosphate dissolved in water were
mixed with p-terphenyl dissolved in dioxane. It
was found that with the exciting light at 300 mu,
tiboflavin and riboflavin-5-phosphate were effec-
tive acceptors of excitation energy from p-ter-
phenyl. Thyroxine dissolved in absolute alcohol
was added to the mixture of p-terphenyl and
ANPO in toluene. There was a definite inhibition
of p-terphenyl fluorescence. At high concentra-
tions of ANPO, there was further augmentation
of the fluorescence of the p-terphenyl enhanced
fluorescence of ANPO while at low concentrations
of ANPO, there was inhibition.

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

427

1391. Morphine antidiuresis in rats with hy-
pothalamic lesions. RopertT GEORGE* AND
E. Leona Way. Dept. of Pharmacology and
Exptl. Therapeutics, Univ. of California Med.
Ctr., San Francisco.

Several investigators have reported that the
antidiuretic effect of morphine is mediated
through the stimulation of the supraoptic nuclei
of the hypothalamus which in turn results in a re-
lease of antidiuretic hormone (ADH). However, it
has also been reported that the mechanism of mor-
phine antidiuresis is a dual one in which a) the
hypothalamus is stimulated to release ADH and
b) an alteration in renal hemodynamics occurs. In
this study lesions were placed in the hypothalamus
to produce diabetes insipidus. To test the hy-
pothesis that the antidiuretic effect of morphine
is mediated solely via the supraoptico-hypo-
physeal tract graded doses of morphine were in-
jected into normal rats and rats with diabetes
insipidus. Antidiuresis was determined by the per-
centage of water load excreted in 120 min. It was
observed that normal and diabetic water-loaded
animals excreted 70.8 and 67.4%, respectively.
Large doses of morphine (10.0, 2.5 and 1.0 mg/kg)
produced a marked antidiuretic effect in both
groups. Smaller doses of morphine (0.1 and 0.05
mg/kg), although producing antidiuresis in nor-
mal animals, failed to inhibit diuresis in animals
with lesions. It would appear from our data that
interruption of the supraoptico-hypophyseal
tract, as evidenced by the diabetes insipidus which
follows, does not completely abolish the antidiu-
retic effect of morphine in rats. These results sug-
gest that the antidiuretic effect of morphine is
produced by more than one mechanism. (Sup-
ported, in part, by a grant from the Natl. Inst. of
Health, Bethesda, Md.)

1392. Mechanism of action of 3,6-bis(3-di-
ethylaminopropoxy) pyridazine bis-meth-
iodide, a long acting neuromuscular block-
ing agent. R. M. GesLer* anp JaMEs O. Hoppe.
Sterling-Winthrop Research Inst., Rensselaer,
be oss
Preliminary studies on the neuromuscular

blocking properties of 3,6-bis (3-diethylamino-

propoxy) pyridazine bis-methiodide revealed a

pattern of action in the cat sciatic-gastrocnemius

preparation and in the unanesthetized animal
which clearly differed from that of either d-tubo-
curarine or decamethonium and which suggested
that a different mechanism of action may be in-
volved. Further studies have shown that, 1) the
production of neuromuscular blockade by this
compound in the cat sciatic-gastrocnemius prepa-
ration is dependent upon the frequency of nerve
stimulation; this blockade is not the result of con-
traction fatigue and its slow onset is not due to

428

delayed access to its site of action; 2) this com-
pound does not antagonize acetyl choline on the
frog rectus abdominis preparation, but 3) it pro-
duces a flaccid paralysis in the pigeon, and 4)
blockade in the cat sciatic-gastrocnemius prepa-
ration and head drop in the rabbit are antagonized
by neostigmine. These findings are not easily ex-
plainable in terms of either a d-tubocurarine- or
decamethonium-like mode of action. Yet the com-
pound apparently acts at the post junctional mem-
brane since it reduces the response of the cat
gastrocnemius to close intra-arterial acetyl cho-
line, and it has weak excitatory effects on the frog
rectus abdominis and the denervated cat gastroc-
nemius, as well as an anticurare action in the cat
nerve-muscle preparation.

1393. Biosynthesis of 5-hydroxytryptamine
(serotonin, enteramine). NicHOLAS J. GIaR-
MAN. Dept. of Pharmacology, Yale Univ., New
Haven, Conn.

The activity of the enzyme, 5-hydroxytrypto-
phane decarboxylase, which forms 5-hydroxy-
tryptamine (HT), was studied by incubating tis-
sue homogenates with appropriate amounts of the
substrate (5-hydroxytryptophane) and estimating
the HT by bioassay. It is a rapidly functioning
enzyme which occurs exclusively in the nonpar-
ticulate fraction of ultracentrifuged cells. Kidney,
liver and gastrointestinal tract showed a high ca-
pacity for forming HT, while spleen, platelets and
bone marrow had little or no activity. These re-
sults eliminate the last 3 tissues as possible sites
of formation of HT in the organism. Sympathetic
ganglia and some parts of the brain showed con-
siderable activity, an observation which supports
the theory that HT may play a physiological role
in some nervous tissues. Despite several interest-
ing similarities, there are many differences be-
tween the distribution of the enzyme and that of
the HT which can be extracted from tissues. The
enzyme in nervous tissues was inhibited by an
excess of substrate, but in various other tissues
such inhibition was not observed.

1394. Myecardial activity of some sympatho-
mimetic amines. JEROME M. GLASSMAN,*
Rosert W. HEATON* AND JOSEPH SEIFTER.
Wyeth Inst. for Med. Research, Radnor, Pa.

The activity of the N-methyl-N-(p-hydroxy-
phenyl), N-methyl-N- (omega-hydroxy-omega-
phenyl), omega-chloro-omega-pheny]-tertiary-
butylamines, as well as 1-(3-oxypheny]l)-1-oxy-2-
ethylamino-ethane (Effortil) was determined on
the dynamic and hypodynamic isolated frog heart
utilizing a modification of the Straub technique,
and was compared to that of the related mephen-
termine (Wyamine: N-methyl-omega-pheny]-ter-
tiary-butylamine). Alterations in the mephenter-
mine molecule as demonstrated by the above

FEDERATION PROCEEDINGS

Volume 16

amines resulted in compounds with lower cardio-
toxicity than methentermine. However, only Ef-
fortil was able to produce a positive inotropic
effect similar to that of mephentermine, but it was
less potent than the latter in this respect. The
other amines appeared to be myocardial depres-
sants. In studies with hearts made to beat arrhyth-
mically by perfusion with epinephrine, only
mephentermine could block the arrhythmia and
could convert the epinephrine induced arrhythmia
to normal rhythm.

1395. Pharmacology of d-lysergic acid mor-
pholide. Joun H. Gocrerty* anp J. M. Dit,
Dept. of Pharmacology, School of Medicine, Univ,
of Washington, Seattle.
d-Lysergic acid morpholide (LSM) differs from

d-lysergic acid diethylamide (LSD) in that the di-

ethylamine side chain is replaced by a morpholine

group. The LD» for LSM was found to be 55 mg/kg
intravenously in mice. Characteristic responses of
excitability and hyperreflexia with recovery in
about $ hr. were produced by 0.8 mg/kg i.v. Larger
doses of 3.2 mg/kg i.v. produced hyperactivity
with recovery in about 45 min. These characteris-
tic responses were intensified and prolonged as the
dose was increased. The smallest dose producing
death was 50 mg/kg. Immediately preceding death
there was respiratory failure followed by convul-
sions presumably due to anoxia. In rabbits dose
levels of 60 ug/kgi.v. produced a rise in tempera-
ture of 1.5°C-2.0°C. With larger doses of 100 ug/kg

i.v., there was, in addition, hyperventilation and

hyperexcitability. Death was produced in rabbits

with 400 ug/kg i.v. In cats a characteristic ‘rage’

response was produced with doses of 600 ug/kg i.v.

This response was immediate and lasted about 20

min. There was salivation, pupillary dilatation

and pilo-erection. Responses were recorded by
motion pictures and compared to those of LSD,

Gross similarities of response were observed, but

with LSM there appeared to be a fear element ac-

companying the ‘rage,’ the cats using every oppor-

tunity to ‘escape.’ With a dose of 1.6 mg/kg i.v.

the characteristic behavior just described was less-

ened as a depression phase developed, marked by
motor incoordination, depressed reflexes and gen-
eral lethargic behavior. In cats doses of 3.2 mg/kg

i.v. did not produce death.

1396. Analgesic effectiveness of orally admin:

~~ Se ae eee

col

fus
ley
op

cor
bul
wel

istered heptacyclazine in man. Mauvrici

Go.sey,* Pau. Lerrer,* ArtHuR J. Gross:

MAN* AND RosertT C. BatTTerRMAN. Dept. df

Medicine, New York Med. College-Metropolitan

Med. Cir., New York City.

The establishment of dosage, effectiveness and
safety of heptacyclazine (1-methyl-4-phenyl-4
carbethoxy hexamethylenamine hydrochlorid
(Federation Proc. 1955) allowed further evaluatiot

ume 16

cardio-
nly Ef-
otropie
b it was
t. The
depres-
rrhyth-
, only
lia and
ythmia

| mor-
DILLE,
2, Univ.

rs from
the di-
pholine
) mg/kg
onses of
very in
. Larger
activity
acteris-
d as the
oducing
g death
convul-
its dose
2mpera-
0 wg /kg
ion and
rabbits
¢ ‘rage’
(/kg iv.
bout 20
latation
‘ded by
of LSD.
red, but
nent ac-
y oppor-
/kg i.¥.
vas less-
rked by
ind gen-

2 mg/kg

admin:

March 1956

of clinical usefulness for relief of pain secondary
to a wide variety of medical and surgical condi-
tions. A bed-ridden group of 118 patients pre-
sented a satisfactory analgesic response to 50 or
100 mg q.i.d. orally in 58% of 139 trials. Patients
with musculo-skeletal conditions, early metastasis
from various neoplasms and neurologic type -of
pains noted the greatest relief. Eighty percent
of an additional 35 post-partum patients had satis-
factory analgesia. In 85 ambulatory patients 50
mg q.i.d. resulted in relief of pain in 64 patients
(75%). Untoward reactions were insignificant.
Nausea occurred in only 4 patients (2%) out of 238
patients treated. Dizziness occurred once. Hepta-
cyclazine satisfies to a high degree the criteria for
a moderately potent analgesic administered
orally.

1397. Biochemical response to trauma. II.
morphine conjugation. LEo R. GoLpBaum,
Irvine Gray,* Ricuarp A. Rinx,* Rouanp R.
RUECKERT,* AND ALVIN 8. OSTASHEVER.* Dept.
of Biochemistry, Walter Reed Army Inst. of Re-
search, Washington, D. C.

The effect of mechanical trauma on morphine
conjugation has been investigated using 2 in vitro
systems: 1) rat liver mince and 2) rabbit liver per-
fusion. Livers, removed from male Sprague-Daw-
ley rats, were minced by forcing them through the
openings of a household-type garlic press. 400 mg
of mince were put into 50 ml Erlenmeyer flasks
containing 5 ml of Krebs-Henselheit bicarbonate
buffer, pH 7.4, and 250 wg of morphine. The flasks
were placed in a Dubnoff shaker, gassed with 95%
02, 5% COz and shaken for 2 hr. Morphine was
determined by the UV spectrophotometric proce-
dure of Goldbaum. Rats were tumbled 600 turns
in a Noble-Collip drum (90-100% mortality). The
mince made from livers removed immediately
after tumbling, conjugated 60.7% of the morphine,
control liver minces, 76.7%. The difference was
significant, P < 0.001. This inhibition had disap-
peared from the livers of animals that were killed
l hr. after the trauma. In the perfusion experi-
ments, livers were removed from female rabbits
that had been hemorrhaged to 40 mm Hg and held
there for 90 min. The average half-life for the con-
jugation of morphine by livers from control rab-
bits was 3.7 min.; from hemorrhaged rabbits 11.2
min. This difference was significant, P < 0.001.
After being perfused for 1 hr., the half-life in the

TAURICE traumatized liver had returned to normal.

Gross:
Dept. a
‘opolitan

ress and
henyl-4
chlori

aluation

1398. Effects of total epidural sympathetic
block on cardiac arrhythmias developing
during hypothermia. Lron I. GoLDBERG AND
Kurt F. Scumipt.* Anesthesia Lab. of Harvard
Med. School, at Massachusetts General Hosp.,
Boston.

The effects of hypothermia were studied in con-

E XUM

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

429

trol dogs and in dogs in which a total sympathetic
block was produced and maintained by the epi-
dural injection of 0.45% procaine HCl solution.
All animals were anesthetized with i.v. thiopental
sodium and were mechanically ventilated with
100% oxygen by means of an Emerson Resuscita-
tor. Hypothermia was produced extracorporeally
by means of a veno-venous shunt and was con-
tinued until ventricular fibrillation or cardiac ar-
rest occurred. Femoral arterial pressure and elec-
trocardiograms were recorded frequently in each
experiment. In the control series of animals,
0.85% saline solution was injected epidurally in-
stead of procaine solution. It was found that the
animals blocked with procaine developed arrhyth-
mias at significantly higher temperatures than
animals without such block. In other hypothermia
experiments with blocked and nonblocked dogs,
right and left auricular, pulmonary and femoral
arterial pressures, heart contractile force (by the
strain gauge technic, Proc. Soc. Exper. Biol. &
Med. 84: 263, 1953) and electrocardiograms were
synchronously recorded at frequent intervals by
means of a Sanborn multichannel oscillograph.
Results of these studies demonstrated that in the
majority of experiments, arrhythmias developed
before myocardial contractility was depressed.
The cardiovascular effects of administration of
epinephrine, norepinephrine, hexamethonium and
tetramethylammonium and of adrenalectomy were
also studied in similar hypothermic experiments
and results will be described. (Supported (in part)
by funds provided under contract with the USAF
School of Aviation Medicine, Randolph Field,
Texas.)

1399. Kidney homotransplantation in dogs.
Howarp GoopMaN,* JEROME Kay,* RosBerr
GAERTNER* AND JAMES Baxter. Natl. Heart
Inst., Bethesda, Md.

A kidney transplanted from a donor dog to the
neck of a recipient dog functions for about 5 days
(Dempster; Hume). If, after failure of function of
the 1st homotransplanted kidney, the 2nd kidney
from the same donor is transplanted to the same
recipient, the 2nd homotransplanted kidney func-
tions for less than 24 hr. Presumably this is due to
development in the recipient of an actively ac-
quired immunity to donor tissue. We have at-
tempted to sensitize recipient dogs to donor tissue
by implanting a piece of donor kidney in the rectus
muscle of the recipient dog, leaving the remainder
of the donor kidney available for preparation of
extracts. Two weeks later the other donor kidney
was transplanted to the neck of the recipient dog.
In 5 out of 6 dogs, these kidneys functioned less
than 24-hr. (in contrast to 4-5 days in our con-
trols), indicating that intramuscular implantation
of kidney tissue may serve to sensitize a recipient

430

dog to donor kidney tissue. The effect of admini-s
tration of extracts of trypsin-digested kidney on
the survival of donor kidneys in such sensitized
recipients will be discussed. In addition, we have
carried out studies on the importance of red blood
cell iso-antigens in the immune response to homo-
transplanted tissue. Donor kidneys were found to
function 5 days in 2 recipient dogs even though the
red cells of the donors were agglutinated by sera
of the recipients which apparently contained anti-
D isohemagglutinins (Young). This suggests that
the recipient immune response which damages the
donor kidney is an immune response to antigen or
antigens other than red cell isoagglutinogens.

1400. Comparison of antiarrhythmic and local
anesthetic actions and toxicities of lido-
eaine hydrochloride, its bicycloheptyl and
cyclohexyl analogs. M. L. Gramme,* H. Le-
HOTzZKY,* R. A. Capmus,* E. A. SteamMunp,* A.
H. CAMPBELL, JR.* AND Go Lu. Johnson & John-
son Research Fndn. and Research Div. of Ethicon,
Inc., New Brunswick, N. J.

In anesthetized, open-chest dogs, auricular ar-
rhythmias were induced by applying pledgets
soaked with 5% methacholine chloride aqueous
solution to the sinus node region and by accom-
panied mechanical pinching of the auricular wall.
As compared with the control, the duration of ar-
rhythmias was shortened 46.5% by 5 mg/kg (i.v.)
of quinidine sulfate. In similar doses, lidocaine hy-
drochloride, N-(2-endobicyclo-(2,2,1)-heptane)-
diethylaminoacetamide hydrochloride (ERL-239),
and N-cyclohexyldiethylaminoacetamide hydro-
chloride (ERL-271) shortened the duration by
55.5%, 80.3% and 24.2%. The relative potencies
were: 1.00, 1.45 and 0.44, respectively. The rel-
ative potencies of intracutaneous anesthesia,
determined in guinea pigs, were: 1.00 (lidocaine
hydrochloride), 0.16 (bicycloheptyl) and 0.07 (ey-
clohexyl). Determination of intraperitoneal LDs59’s
in mice yielded the relative toxicities: 1.00 (lido-
caine hydrochloride), 0.50 (bicyclohepty]), and 0.58
(eyelohexyl). Evidently, the substitution of a bi-
cycloheptyl ring for the dimethylpheny]! ring in
the lidocaine molecule reduces both the local anes-
thetic activity and the toxicity, but preserves or
enhances the antiarrhythmic activity. This adds
to evidence that local anesthetic and antiarrhyth-
mic actions of a compound do not necessarily cor-
relate. In anesthetized dogs with ventricular ar-
rhythmias induced by intravenous ouabain, 20-40
mg/kg (i.v.) of the bicycloheptyl analog either re-
duced the rate of arrhythmias or restored it to
sinus rhythm temporarily.

1401. Analgesic power and toxic effects in man
of dihydrocodeine and dihydroisocodeine
compared with morphine and a placebo.
JoacuIM S. GRAVENSTEIN,* GENE M. Smitu,*

FEDERATION PROCEEDINGS

Volume 16

RayMoNnpD D. Spuire,* James P. Isaacs* aNpb

Henry K. Beecuer. Anesthesia Lab. of Harvard

Med. School at Massachusetts General Hosp.,

Boston.

The analgesic potency of dihydrocodeine bitar-
trate and dihydroisocodeine bitartrate was com-
pared with morphine phosphate in patients with
postoperative wound pain. The patients received
the experimental drug and the standard dose of
morphine alternately. In normal volunteers objec-
tive and subjective side effects were studied using
equal analgesic doses of the experimental drugs
and morphine. A placebo was inserted. Attention
was focused on respiratory depression, expressed
in terms of minute volume and rate, and changes
in subjective state and mood measured in a psy-
chological evaluation which used a questionnaire
and an adjective check list. Dihydroisocodeine is
similar to dihydrocodeine in analgesic power;
however, its side effect liability is very like that
of morphine in equal analgesic dose. The out-
standing result of this study was the finding that
dihydrocodeine in the optimal dose of 30 mg pro-
duced little if any respiratory depression and few
if any undesirable psychological effects, i.e., it
was similar to a placebo as far as side effects were
concerned. The same dose proved to be an effec-
tive analgesic, comparable to morphine 10 mg.
The duration of action of dihydrocodeine is some-
what shorter than that of morphine. This of little
clinical importance in view of the lack of side
effects.

1402. Anticonvulsant activity of 5 - acetyl-
imino - 4 - methyl - A? - 1,3,4 - thiadiaz-
oline-2-sulfonamide, (CL 13,912), a new
carbonic anhydrase inhibitor. W. D. Gray,
T. H. Maren, G. M. Stsson* ann F. H. Smitru.*
Exptl. Therapeutics Section, Research Div.,
American Cyanamid Co., Pearl River, N. Y. and
Stamford, Conn.

Elimination of the dissociable hydrogen from
the carboxamide group of Diamox acetazoleamide
led to the synthesis of CL 13,912, the pharmacol-
ogy of which is described elsewhere (G. M. Stsson
et al. Federation Proc., this issue). CL 13,912 has
3 to 4 times the anticonvulsant activity of aceta-
zoleamide against supramaximal electroshock
seizures in mice. Oral ED59 of the new compound,
CL 13,912, is 18 mg/kg (P 0.05 limits 15-22).
Significant protection remains 8 hr. after dose.
Oral EDs of sodium diphenyl] hydantoin is 13 mg/
kg (P 0.05 limits 12-14). At 5 mg/kg orally CL
13,912 begins to increase current necessary for
maximal seizures in 50% of mice (CS;s0) made
hyponatremic by intraperitoneal administration
of isosmolar glucose. The corresponding figure in
normal mice is 10 mg/kg. At 20 mg/kg in hypo-
natremic mice, CL 13,912 increases CSs from

ume 16
3* AND
larvard
Hosp.,

bitar-
s com-
8 with
ceived
lose of
objec-
1 using
drugs
ention
pressed
_hanges
a psy-
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e that
e out-
ig that
ig pro-
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s were
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some-
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cetyl-
adiaz-
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GRAY,
MITH .*

Div.,
Y. and

. from
‘amide
macol-
SISSON
12 has
aceta-
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ound,
[5-22).
dose.
i3 mg/
ly CL
ry for

made
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rure in

hypo-
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March 1956

8-85 ma, while sodium diphenyl hydantoin in-
creases C85. to > 500 ma. CL 13,912 is inactive in
the subcutaneous Metrazole test. Plasma levels
following oral and intravenous administration of
CL 13,912 indicate complete absorption from the
mouse gastro-intestinal tract. Plasma half-time
in mice is 1.1 compared with 0.2 hr. for acetazole-
amide. Five minutes after intravenous administra-
tion of CL 13,912 at 20 mg/kg the concentration
of inhibitor in mouse brain, corrected for trapped
blood, was 2.7y7/gm; that following acetazole-
amide was zero. Peak concentration of inhibitor
in brain is achieved within minutes after intra-
venous administration of CL 13,912, and precedes
peak anticonvulsant activity by 1-2 hr. Since
concentrations of inhibitor in brain are relatively
low at time of peak protection, the data suggest
that systemic or CNS inhibition of carbonic
anhydrase initiates events which lead to the anti-
convulsant effect.

1403. Modification of CNS drug action by
anticholinesterases. VERNON A. GREEN* AND
JoHN E. Davis. Univ. of Texas, College of
Pharmacy, Austin.

This study is concerned with the effects of the
injection of physostigmine, neostigmine, or
D.F.P. intraperitoneally into white mice prior
to the intravenous injection of strychnine, pentyl-
enetetrazol, phenobarbital sodium, or thiopental
sodium. Physostigmine (0.4 ng/gm) given 15 min.
prior to strychnine rendered a subconvulsive dose
of strychnine (0.25 ug/gm) convulsive and fatal,
(30 animals). Neostigmine in the same dose had
the same effect as physostigmine in 20 animals
tested. D.F.P. (2 mg/kg) administered 2 hr.
before strychnine, caused convulsions in 10 ani-
mals tested, but none died. The anticholinester-
ases alone caused no effects and their synergistic
actions with strychnine were not blocked by
atropine (up to 1 mg/gm). In experiments with
pentylenetetrazol, it was found that prior ad-
ministration of physostigmine increased the con-
vulsive activity of metrazol (12.5 and 10 mg/gm)
and also extended the duration of the convulsive
seizures. Forty experimental animals were given
phenobarbital sodium (160 mg/kg) after prior
injection of anticholinesterases. All 3 anticholin-
esterases were effective in decreasing the time
lapse until onset of anesthesia to less than half
that required for a similar control group. In
experiments using thiopental sodium (35 mg/kg)
it was found that premedication with physostig-
mine prolonged the mean duration of anesthesia
about 4-fold. When a lower dose of thiopental
(10 mg/kg) was used, no anesthesia occurred in
30 control animals, but brief anesthesia almost
invariably developed in experimental animals
premedicated with physostigmine, neostigmine
or D.F.P.

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

431

1404. Method for assay of cathartic agents in
ambulant patients. T. GREINER, H. Go.p,
I. Bross,* L. WarsHaw* anv N. Kwirt.* Depis.
of Pharmacology and of Public Health and Pre-
ventive Medicine, Cornell Univ. Med. College;
Cardiovascular Research Unit, Beth Israel Hosp.,
and Cardiology Service, Hosp. for Joint Diseases.
New York City.

Few studies of cathartic agents have utilized
the principles of clinical pharmacology, double-
blind evaluation in comparison with inert agents
and proper randomization. Cathartics have gener-
ally been administered to the special populations
incarcerated in nursing homes and have relied on
observations by the attendant or nurse to supply
information. There is need of extending the
methods to include the constipated ambulant
population and also to provide a system for de-
tecting minimal changes in the bowel habits of
normal subjects as induced by special food mate-
rials. A daily report card was designed for self-
reporting by the subject. Each night the subjects
recorded the number and consistency of their
bowel movements. At clinic visit any judgment
necessary about the dosage regimen or adequacy
of report was made in the double blind state.
There were always 3 materials in a comparison:
thé placebo, a standard and the unknown. Analysis
of variance was used to demonstrate the sensi-
tivity of the method to detect small doses of
cascara sagrada in normal subjects and standard
doses of cascara in constipated subjects. Changes
in the number of bowel movements did not always
accompany changes in the consistency scale. The
effectiveness of an extract of bran was tested in
constipated and nonconstipated subjects.

1405. Influence of various substances on the
uptake of epinephrine by the liver. ERNEST
C. GriEsEMER* AND J. A. Wetts. Dept. of
Pharmacology, Northwestern Univ. Med. School,
Chicago, Ill.

It has been shown by several investigators that
various responses to epinephrine are much greater
following injection into systemic veins than
following injection into the portal vein. The intra-
portal to intra systemic ratio of equieffective
doses has been shown to be about 4 to 1. Little
evidence exists as to the quantitative nature of
this phenomenon of liver uptake but it has been
claimed that certain amidines and methylene blue
are capable of reducing it. In the present studies
it was shown that the amount of epinephrine
apparently removed by the liver is a linear fune-
tion of the amount presented to it. 75 to 80% is
removed by the liver on a single passage. It has
been postulated that epinephrine potentiation
by various agents may involve an interference
with the tissue uptake of epinephrine. It was
thus of interest to determine the influence of

432 FEDERATION
potentiating agents on the liver uptake. Cocaine
was shown to shift the intrajugular and the intra-
portal dose response curves by an equivalent
amount and, thus, fail to alter the slope of the
linear relationship between the amount of epi-
nephrine presented to the liver and the amount
removed by it. On the other hand, choline-p-toly]
ether significantly altered this slope, thereby
reducing the percentage uptake by the liver. The
latter substance, as well as the previously men-
tioned amidines and methylene blue are mono-
amine oxidase inhibitors. Marsilid, a potent
monoamine oxidase inhibitor, failed to influence
the uptake of epinephrine by liver.

1406. Effects of acetazoleamide (Diamox)
upon blood sugar of normal and diabetic
patients. ARTHUR J. GRossMAN,* Nina Fiau-
ROVSKY* AND Rospert C. BaTTeERMAN. Dept. of
Medicine, New York Med. College-Metropolitan
Med. Ctr., New York City.

The influence of acetazoleamide (Diamox) upon
fasting blood sugar was studied in 20 diabetic and
20 normal subjects. Half of each group were either
given 500 mg of acetazoleamide orally and fasting
blood sugars determined (method of Folin-Wu)
3 hr. later or followed without drug administra-
tion for a similar period. One week later, to
eliminate biologic variation, the subjects were
restudied but the procedure reversed. Both the
normal and diabetic group of patients with and
without acetazoleamide revealed no significant
alteration of the fasting blood sugar. Acetazole-
amide was given in doses of 500 mg daily for a
3-wk. period to 3 normal and 3 diabetic patients.
Fasting blood sugars (method of Somogyi) were
determined at weekly intervals. A similar group
was followed without drug administration. No
significant alteration of the fasting blood sugar
was noted after chronic administration of acet-
azoleamide in diabetic and normal subjects.
Effects upon sugar tolerance curve will be pre-
sented.

1407. Clinical experiences during anesthesia
with a new ultra-short-acting barbiturate.
CuHartes M. Gruser, Jr., Virait K. SToEtt-
1nG,* M. L. Hicks* anp SamuEet Dovueury.*
Lilly Lab. for Clin. Research and Dept. of Anes-
thestology, Indiana Univ. School of Medicine,
Indianapolis.

Compound 22451 was administered intermit-
tently (1.0% solution) or by continuous drip
(0.1% solution) intravenously to 650 patients for
induction and/or maintenance of anesthesia. No
special selection was made as to the condition,
age or sex of the patient; the other anesthetics to
be used; the operation to be performed; or the
use of skeletal muscle relaxants. Trained anes-
thesiologists and residents in anesthesiology used

PROCEEDINGS

Volume 1§

22451 whenever they thought an intravenous
anesthetic was indicated. Preanesthetic medica-
tion usually consisted of a barbiturate or narcoti¢
and atropine or scopolamine. For purposes of
comparison, old anesthesia records from this same
hospital were used. The doses of Compound 22451,
Pentothal and Surital used for induction were,
respectively, 54.4+18.8, 156.5+69.8 and 226.44
92.5 mg. During nitrous oxide and oxygen anes-
thesia, 57.4, 58.0 and 153.0 mg, respectively, of
22451, Pentothal and Surital were given per hour
by intermittent injection; 94.9 mg of 22451/hr.
were given by continuous drip. Generalized
muscular tremors were seen frequently, but were
considered so unimportant that the anesthesiol-
ogists had to be encouraged to report them. Seri-
ous convulsions were not seen although on 2
occasions the medication was changed because
of skeletal muscle activity. The onset of anes-
thesia was more rapid; the duration of a given
dose shorter; and the patients, on awakening, were
surprisingly alert with Compound 22451.

1408. Intracellular distribution of acetyl.
choline and cholineacetylase in human
placental tissue. Paut HaGeEn (introduced
by D. BonnycastLE). Dept. of Pharmacology,
Yale Univ., New Haven, Conn.

Three pharmacologically active bases present
in mammalian tissues, epinephrine (BLASCHKO
AND Wetcu, Arch. exper. Path. u Pharmakol.
219: 17, 1953), histamine (Haamn, Brit. J. Phar-
macol. 9: 100, 1954), and 5-hydroxytryptamine
(BLASCHKO, personal communication), are con-
tained within intracellular particles in tissues
rich in these compounds. The intracellular dis-
tribution of another pharmacologically active
base, acetylcholine (ACh) has now been investi-
gated. The tissue used for this study was human
placenta, a tissue known to be rich in this base
(CHANG AND GappuM, J. Physiol. 79: 255, 1983).
Fresh placental tissue was fractionated as pre-
viously described (BLAScHKO, HAGEN AND WELCH,
J. Physiol. 129: 27, 1955). The ACh content of the
fractions was determined by assay on the frog
rectus. The mean values for ACh obtained from
3 experiments, expressed as ng ACh/gm of original
tissue, were: large granule fraction, 0; microsome
fraction, 0; high-speed supernatant, 13. The
cholineacetylase activity of the fractions was also
determined. The mean results of the experiments,
expressed as yg ACh synthesized per gram original
tissue per hour, are: large granule fraction, 1;
microsome fraction, 1; high-speed supernatant, 80.
It is concluded that both ACh and the enzyme
which synthesizes it are located in the nonparticu-
late cytoplasm of the cell. Thus ACh differs in its
intracellular localization from the other common
pharmacologically active bases found in mam-
malian tissues.

Th
Ox!

bit
eff
an
20)
pr

lume 1§

avenous
medica-
narcotic
0ses of
his same
id 22451,
mn were,
226.44
on anes-
vely, of
yer hour
2451 /hr.
eralized
yut were
sthesiol-
m. Seri-
h on 2
because
of anes-
a given
ng, were
i.

acetyl-
human
roduced
vacology,

present
LASCHKO
armakol,
!. Phar-
ptamine
ire con-
tissues
lar dis-
active
investi-
; human
his base
9, 1933).
as pre-
WELCH,
it of the
the frog
ed from
original
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(3. The
was also
riments,
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March 1956

1409. Intra-cerebral injection of LSD-25 in
the unanesthetized dog. Tuomas J. Hatey
AND W. G. McCormicx.* Dept. of Medicine,
School of Medicine, Univ. of California at Los
Angeles, Los Angeles.

Using the implanted cannula, described pre-
viously (Circ. Research 3: 103: 1955), lysergic acid
diethylamide (LSD-25) was injected into the 3rd
cerebral ventricle of the unanesthetized dog.
Doses of 1.6, 5 or 10 ug/kg produced the following
symptoms: 1 min.—whining and shaking of the
head; 2 min.—licking the chops, salivation and
atoxia; 3 min.—retching, nausea, emesis, mictura-
tion and tachypnea; 5 min.—all above much
exaggerated; 6 min.—mydriasis with reactive
pupils; recovery occurred after 15-20 min.
Throughout this period the animals appeared
frightened, but there was no impairment of their
ability to follow simple commands or perform
simple tasks. The animals reverted from an adult
behavior pattern to a puppy one resembling in
part the changes observed in humans by Forrer
and Goldner (Arch. Neurol & Psychiat. 65: 581:
1951). Much larger intravenous doses of LSD-25
are required to produce the pharmacological
effects observed after intraventricular injection
of the drug. The most striking effect is the per-
sonality change which occurs after intraventricu-
lar injection of LSD-25. The other symptoms are
in part similar to those observed by Weinberg
and Haley (Circ. Research 3: 103: 1955; Proc.
Soc. Exper. Biol. & Med. 89: 345: 1955; Arch.
internat. pharmacodyn. In press) in dogs receiving
cardiac glycosides, quinidine, procaine amide or
chlorpromazine or by Feldberg and Sherwood
(J. Physiol. 123: 148: 1954) in cats receiving many
chemically and pharmacologically related drugs.
There can be no doubt that intraventricular
injection of drugs in unanesthetized animals
allows a better evaluation to be made of the
central effects of drugs. (Supported by a grant
from Ciba Pharmaceutical Products, Inc.)

1410. Some pharmacological properties of
the quaternary oxazolium compounds.
Tuomas J. Hater, W. G. McCormick* anp
A. M. FriesHer.* Div. of Pharmacology and
Toxicology, Atomic Energy Project, School of
Medicine, Univ. of California at Los Angeles,
Los Angeles.

Lushbaugh et al. (J. Pharmacol. & Exper.
Therap. In press) observed that the quaternary
oxazolium compounds had a poikilothermic effect
in mice and rats and an antipyretic effect in rab-
bits. We have investigated the pharmacological
effects of 2(1-naphthyl)-, 2-(4-methoxypheny]l)-,
and 2-(4-methylpheny])-3-methyl-5-phenyloxa-
zolium-4-toluenesulfonate. All of these compounds
produced a hypotension in chloralose or pento-
barbitalized cats and the response was linear

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

433

over the dosage range 0.5-5 mg/kg. There was
no detectable change in heart rate with these
doses, but at higher doses, cardiac anoxia was
evident because there was an increase in the height
of the T-wave of the ECG. Respiratory rate
decreased slightly and death was the result of
respiratory paralysis coupled with peripheral
vascular collapse. None of these effects were
modified by 2 mg/kg of atropine, 1 mg/kg of
pyrilamine maleate or bilateral vagotomy. The
oxazolium compounds had no ganglionic blocking
or stimulating effects on the superior cervical
ganglion. These compounds showed no spasmo-
lytic or adrenergic blocking properties. However,
they increased the tonus and amplitude of con-
traction of the cat intestine in situ and reduced
the body temperature of these animals as much
as 2°C. The oxazolium compounds did not produce
chromoachryodynia in rats at doses as high as
500 mg/kg and they did not block such responses
induced by methacholine. At concentrations of
1% these compounds had no effect on the rabbit
eye. However, they prevented epinephrine tox-
icity in mice probably by peripheral vasodilata-
tion rather than adrenergic blockade. Upon the
basis of this investigation, it seems probable
that these compounds could be applied in surgical
cases requiring hypothermia. (This article is
based on work performed under Contract No. At-
04-1-GEN-12 between the Atomic Energy Com-
mission and the Univ. of California at Los
Angeles.)

1411. Effect of whole body x-irradiation on
the free amino acid content of rat plasma.
Duane W. Hatiesy* anp JoHN Doutt. U. S.
Air Force Radiation Lab. and Dept. of Pharma-
cology, Univ. of Chicago, Chicago, Il.

Recent studies by Kay and Entenman (Federa-
tion Proc. 13: 520, 1954) and Mefford and Martens
(Science 122: 829, 1955) have demonstrated changes
in the free amino acid excretion patterns of x-irra-
diated rats. The present study was carried out
to determine the effect of x-irradiation on the
free amino acid content of rat plasma using the
ion-exchange method described by Moore and
Stein (J. Biol. Chem. 192: 663, 1951) for the amino
acid separation and ninhydrin for the quantita-
tive estimation of the individual amino acids.
Adult male Sprague Dawley rats were given 800 r
of whole body x-irradiation (radiation factors:
250 KVP, 15 ma, target-skin distance 75 cm, dose
rate 35-37 r/min). Blood was obtained by cardiac
puncture at 1, 2, 3 and 5 days after the x-ray
exposure. Both the control and the irradiated
animals were fasted during the postirradiation
period to eliminate the effects of the radiation-
induced anorexia. The plasma content of serine,
glutamic acid, valine, methionine, lysine, alanine,
threonine, isoleucine and leucine was increased

434

at 24 hr. after the x-ray exposure. The plasma
levels of tryptophan, arginine and aspartic acid
were unchanged and that of glycine was decreased.
These effects were transitory lasting only about
24-48 hr. except for the increases in the amounts
of threonine, isoleucine and leucine and the de-
crease in the quantity of glycine which persisted
throughout the 5-day observation period.

1412. Bronchodilator and _ blood pressure
activities of a series of N-alkyl-o-methoxy
(and o-hydroxy) -8-phenylisopropylamines.
CaLviIn HANNA AND RoBERT L. Driver. Dept.
of Pharmacology, Univ. of Vermont College of
Medicine, Burlington.

Studies on the pharmacology of a series of
N-alkyl-o-methoxy-8-phenylisopropylamines and
N-alkyl-o-hydroxy-8-phenylisopropylamines have
been accomplished. Arterial blood pressure was
recorded in the pentobarbitalized cat and dog.
Bronchodilator activity was studied (in the pento-
barbitalized dog and guinea pig) by comparing
the changes in pressure in the bronchial tree
under intratracheal positive pressure respiration.
Compared to isopropylarterenol, the series of
N-alkyl-o-methoxy-8-phenylisopropylamines were
weak to moderate vasodepressor agents. These
compounds were compared with Orthoxine (N-
methyl-o-methoxy -8-phenylisopropylamine) and
isopropylarterenol in ability to combat histamine
induced increases in pulmonary resistance. The
series of N-alkyl-o-hydroxy-8-phenylisopropyl-
amines were weaker vasodepressor agents than
the corresponding o-methoxy- series of compounds
except for N-methyl-o-hydroxy-8-phenylisopro-
pylamine (EA-183) which had vasopressor activity
similar to that of ephedrine.

1413. Action of furfurylideneacetone and re-
lated compounds on the cardiovascular
system. Mervyn G. HarpinGceE (introduced
by Rosert Dretspacu). Depts. of Pharmacol-
ogy, Schools of Medicine, College of Med. Evange-
lists, Loma Linda, and Stanford Univ., San
Francisco, Calif.

The action of furfurylideneacetone (FFA) on
the heart and blood pressure was observed in
decerebrated and spinal cats and in dogs under
ether anesthesia. Standard pharmacological tech-
niques were employed and these included electro-
cardiograms taken of the heart. Changes in spleen
and intestinal volumes were also recorded. The
direct effect of the drug on excised hearts, coro-
nary arteries and peripheral blood vessels of cats,
rabbits, guinea pigs, rats and frogs was made by
direct perfusion of these tissues. The intravenous
injection of the drug in anesthetized animals
resulted in a sudden and marked drop in blood
pressure associated with engorgement of the
splanchnic vessels and passive contraction of the

FEDERATION PROCEEDINGS

Volume 1§

spleen. In isolated hearts, the cardiac contractility
was depressed, and, in large doses, both rate and
rhythm were disturbed. The perfused coronary
vessels, vessels of rabbit ears and rat hindlegs
were generally dilated. The negative inotropie
action on the heart of FFA in the face of coronary
dilatation suggests interference with oxygen util-
ization. The ECG tracings resembled somewhat
those produced by myocardial anoxia. The respira-
tion of heart slices was depressed by FFA. When
lethal doses of FFA were given dogs, death was
due to cardiac rather than respiratory failure,
Similar effects were observed with the use of
furfural acetate, but this drug is far more toxi¢

1414. Influence of calcium ions on normal
tonus and histamine response of isolated
guinea pig ileum. WiLiraAmM D. HARKNEss,*
LEoN Hurwitz* AND GEOFFREY Wooparb,
Div. of Pharmacology, Food and Drug Admin,
Washington, D. C.

Observations have been made of the influence
of Cat* on the normal tonus and on the response
to histamine of the isolated guinea pig ileum,
Prior to each experiment the ileum was stored for
72-96 hr. at 5°C in Tyrode’s solution. It was then
suspended in a muscle bath arranged for recording
isotonic contractions. The bathing fluid was
Tyrode’s solution adjusted to pH 7.4, maintained
at 37.5°C, but with Catt and Mg*t omitted. As
the Ca** concentration was gradually increased
from 0.0 to 0.01% two different effects were ob-
served. Up to approximately 0.001% Ca** there
was little influence on muscle tonus while the
contractile response to a normal dose of histamine
increased from zero to a maximum. From 0.001%
to 0.01% Ca** there was a marked increase in
tonus whereas the response to histamine was re-
duced moderately. In the presence of 0.001% Ca**
the tonus of the ileum can be increased to the
same degree by adding either pilocarpine or excess
Ca**. The additional contractile response elicited
by histamine added to either system was much
lower when pilocarpine was used to bring about
the increase in tonus. The Ca** effect observed
in this stored preparation is different, in some
respects, from results reported by others in which
fresh tissues and/or slightly different ionic en-
vironments were employed

1415. Cumulative effect of small doses of
reserpine on serotonin in man. BERNARD J.
HAVERBACK,* PARKHURST A. SHORE,* EDWARD
G. Tomicn* anp BERNARD B. Bropie. Natl.
Heart Inst., Bethesda, Md.

It has recently been shown that the adminis-
tration of a single large dose of reserpine (5 mg/
kg) to animals caused an almost complete release
of serotonin from the blood platelets, intestinal
tract and brain. Much smaller doses of reserpine

are
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eserpine

March 1956

are used clinically but are usually administered
for prolonged periods of time. The cumulative
effect of small doses of reserpine on platelet sero-
tonin levels was determined in 8 hypertensive
subjects. Studies in 3 of the subjects showed that
a single dose of 1 mg of reserpine had no appre-
ciable effect on platelet serotonin. However, the
daily administration of 1 mg of reserpine for
10 days reduced platelet serotonin by approxi-
mately 95%. Following cessation of reserpine
administration, a number of days elapsed before
platelet serotonin returned to normal levels. No
serotonin could be detected in platelets of 5 hyper-
tensive patients who had been on a reserpine
dosage of 0.5-1.5 mg daily for 3 months. The daily
administration of reserpine in a dosage of
0.015 mg/kg in rabbits likewise resulted in a pro-
gressive decline of platelet serotonin. The brain
serotonin levels in these animals also decreased
but to a lesser degree. The serotonin levels in the
intestine showed no significant change. These
findings indicate that reserpine incapacitates the
serotonin-binding sites in platelets and brain in a
cumulative manner.

1416. Eosinopenic response to cortisone, his-
tamine and epinephrine and prevention of
this change by tripelennamine. Harry W.
Hays AND VirainrA L. ZARATzIAN.* Wayne
Univ. College of Medicine, Detroit, Mich.
Eosinophil counts were taken at 3 hr. intervals

in dogs treated with 150 ug/kg of histamine di-
phosphate, 5.0 mg/kg of cortisone acetate,
30 wg/kg of epinephrine hydrochloride, 1.0 or
3.0 mg/kg of tripelennamine hydrochloride, and
10% sodium chloride. These values were compared
with the changes in normal untreated dogs. It
was found that cortisone, histamine and epi-
nephrine produced a marked eosinopenia; while
tripelennamine and sodium chloride produced
varying degrees of eosinophilia. The onset and
intensity of response varied with the compound
used and was a function of the dose. The response
to cortisone was the greatest while the least
potent was epinephrine. The antihistamine agent,
tripelennamine, when given prior to the adminis-
tration of the eosinopenic agents, prevented
changes in circulating eosinophils due to cortisone
and histamine, but did not block the epinephrine-
induced eosinopenia. The results of this study
would suggest that drug-induced eosinopenia is
the result of the endogenous production and
release of a substance which brings about changes
in eosinophils. This substance is probably hist-
amine. It would also appear that the eosinophils
are not destroyed but are redistributed to other
parts of the body, particularly the reticulo-
endothelial system.

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

435

1417. Respiratory response of the dog to
sodium selenite. Max Hernricu anv D. M.
MacCanon (introduced by F. E. KeEtsry).
Dept. of Physiology and Pharmacology, Univ.
of South Dakota Med. School, Vermillion.
Death from selenium poisoning is commonly

believed to result from respiratory failure. How-

ever, we have observed respiratory stimulation
in unanesthetized dogs and rabbits. The respira-
tory responses to sodium selenite have been
studied further in pentobarbitalized dogs. The
intravenous threshold dose for respiratory stimu-
lation approximates 0.2 mg/kg sodium selenite.
Following threshold doses the respiratory rate
gradually increases. Larger doses result in a rapid
increase to a maximum rate in about 5 min. Al-
though the amplitude of respiration may increase
or decrease, depending upon the respiratory rate,
the minute volume increases in all cases. Func-
tional circulatory collapse precedes the sudden
onset of apnea seen after lethal doses. Oxygen
consumption is either unchanged or slightly de-
pressed after low doses of selenite. The
temporarily increased consumption following
high doses is followed by depression. Oxygen
consumption is negligible shortly before apnea.

Collection of respiratory tract fluid begins shortly

before apnea when 4 mg/kg sodium selenite is

administered. The fluid is mucoid, may be pink
or red, and contains albumin in approximately
the same concentration found in the blood plasma

(based on the concentration of RISA). In many

dogs the fluid represents 1% of the body weight

(approx. 10% of the blood volume). Results

obtained following vagotomy and/or carotid

denervation indicate that both are involved in
the respiratory stimulation.

1418. Therapy of cyanide poisoning. S.
Ricuarp Hersey AND J. PALMER SAUNDERS
(introduced by J. H. Wits). Pharmacology
Branch, Chemical Corps Med. Labs., Md.
Among compounds suggested for the treatment

of cyanide poisoning are NO, NO. + 8:0;7,

p-aminopropiophenone (PAPP) and _ chloroco-

balamin. To assess quantitatively the therapeutic
values of these compounds, tracheotomized cats
lightly anesthetized with pentobarbital were
given 2 LDw’s (3.5 mg/kg) of NaCN intrave-
nously. At cessation of spontaneous respiration,
artificial respiration was instituted at the rate of
25/min. Therapeutic compounds, if administered,
were given i.v. exactly 5 min. after the CN- in-
jection. Initiation of recovery was judged by a)
appearance of attempts at spontaneous respira-
tion and 6) return of higher CNS (wink reflex)
activity. When no therapeutic compounds were
administered, 3 out of 5 animals survived but
with no return of spontaneous respiration or wink

436

reflex. When 5 mg/kg NO2 was given with 375 mg
8.0;7, respiratory efforts were made within 23 min.
and the wink reflex returned within 35 min. When
8.037 was given alone, 1 out of 3 animals survived;
when NO. was given alone, 0 out of 4 animals
survived. When the NO; dose was increased to
10 mg/kg, all animals initiated respiratory efforts
within 12 min. anc the wink reflex returned
within 26 min. The eddition of 8:0;7 at this dose
of NO2 appeared to delay recovery; 0.5 mg/kg
PAPP produced recovery in all animals, with
respiratory attempts within 33 min. and return of
the wink reflex within 48 min. Chlorocobalamin in
a dose of 100 mg/kg gave return of respiratory
activity at 21 min. and of the wink reflex in
43 min. All animals showing return of spontaneous
respiratory efforts and of the wink reflex even-
tually breathed alone and survived.

1419. Effects of meprobamate (Miltown),
chlorpromazine, and reserpine on behavior
in the monkey. CuHartes D. HENDLEY,
Tuomas E. Lynes* anv F. M. Bercer. Wallace
Labs., New Brunswick, N. J.

Meprobamate has been shown to have a re-
markable tranquilizing effect in man and in the
monkey. In the present study, it was found that
at an appropriate oral dose of meprobamate
(250-400 mg/kg) hostile and aggressive rhesus
monkeys could be tamed to a point at which they
showed no hostility toward the experimenters,
and very little fear, while apparently retaining
alertness to sensory stimuli, good appetite and
full awareness of the environment. Some ataxia
and muscular weakness was seen, particularly in
the hind legs. Tactile placing reflexes were abol-
ished while visual placing reactions remained
normal, as did other reflexes. The maximum effect
of meprobamate is reached about 3 hr. after oral
administration. Some recovery is evident at 5
or 6 hr., with return to normal at 12-20 hr. At a
daily oral dose of 250-275 mg/kg for 5 days, there
is no evidence of accumulation of the drug or of
the development of tolerance. With reserpine,
25 and 30 mg/kg per os, all monkeys became im-
mobile for long periods, apparently catatonic,
and showed very little reactivity to stimuli.
Their eyes were generally closed. However, if the
animal was moved bodily, it often became aroused
and might attack and bite the experimenter with
all indications of normal motor ability. With
chlorpromazine, 20-30 mg/kg, the animals first
seemed to lose their fear of the experimenter while
remaining hostile. Later they became very quiet,
seemingly ‘catatonic,’ but awake. They were
usually unresponsive to tactile or other stimula-
tion but would sometimes attack on very slight
provocation. No motor deficit was observed.
With both chlorpromazine and reserpine, the
monkeys appeared to be much more insulated

FEDERATION PROCEEDINGS

Volume 16

from their environment than with meprobamate
while they were not as reliably tamed.

1420. Paper chromatography and separation
of chlorophyll derivatives. M. J. HENDRICK-
SON AND R. R. BeRvueErFry (introduced by A. R.
McIntyre). Univ. of Nebraska College of Medi-
cine, Omaha.

The exhibit depicts the progressive purification
by circular paper chromatography of crude leaf
extract to the chromatographically pure ho-
mogeneous material. This material has a marked
cardiotropic action, and kymographic and elec-
tromyographic tracings are shown illustrating
the effect of the active material on the
hypodynamic heart.

1421. Effects of anesthesia on intermediary
earbohydrate metabolism. Dorotuy UH.
HENNEMAN* AND JOHN P. BUNKER. Anesthesia
Lab., Harvard Med. School at Massachusetts
General Hosp., Boston.

General anesthesia is accompanied by altera-
tions in blood concentrations of intermediary car-
bohydrate metabolites and inorganic phosphorus
which may reflect endocrine and circulatory
disturbances as well as the effects of anesthesia
on cellular metabolism. The relative importance
of these factors has been evaluated in man by
comparing the effects of ether and thiopental
anesthesia, with and without an additional meta-
bolic load (intravenous glucose). Of principal
interest were consistant increases in total serum
inorganic phosphorus during ether, as previously
reported by Foldes, and also during thiopental
anesthesia. The administration of glucose during
anesthesia produced an even greater rise in in-
organic phosphorus instead of the expected fall.
Hyperglycemia was observed as expected and may
well be secondary to impairment in phosphorylat-
ing mechanisms. The glycolytic and oxidative
phases of carbohydrate breakdown, as reflected
by serum lactate and pyruvate, and by serum
citrate and alphaketoglutarate, were undisturbed
during thiopental anesthesia, with and without
intravenous glucose. On the other hand, during
ether anesthesia serum lactate and pyruvate rose
progressively, but Brewster’s demonstration that
a total sympathetic block prevents elevation of
lactate and pyruvate in the etherized dog strongly
suggests that this is secondary to reflex release of
epinephrine rather than to primary depression
of glucose oxidation. True primary depression of
glucose oxidation, as produced by hypothermia,
was accompanied by a fall in serum inorganic
phosphorus, a distinctly different metabolic
response than observed during ether or thiopental
anesthesia. These findings in man are consistent
with reports by Bain and MclIlwain of in vitro
and in vivo effects of barbiturate anesthesia in

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1423

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March 1956

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

437

animals;, namely, an uncoupling of oxidative-2) potentiating action of both is blocked by

phosphorylation without interference in the oxida-
tive pathways of carbohydrate metabolism.

1422. Local anti-inflammatory activities of
some 9 a-halo derivatives of adrenal
steroids. L. G. HeErRsHBERGER,* D. W.
CatHoun* anv V. A. Dritu. Div. of Biological
Research, G. D. Searle & Co., Chicago, Ill.
Granulomas induced by steroid-treated cotton

pellets have been used to evaluate local anti-
inflammatory activity particularly of long-acting
steroids. Adrenalectomized male rats were used.
Cotton pellets weighing 5-8 mg were impregnated
with the appropriate steroid in acetone, which
was readily removed by evaporation. On the day
following adrenalectomy, 2 control and 2 im-
pregnated pellets were implanted in each rat
through small incisions into the loose subcu-
taneous connective tissue. Six days after implanta-
tion, the rats were killed and the granulomas were
dissected out, dried to constant weight and net
tissue weights determined. A bio-assay was per-
formed using a standard (cortisol acetate) and
4 related steroids of unknown potency. Three dose
levels were included for each material, with 2 rats
caged together per dose level for unknowns and
4 animals for the standard. The design was repli-
cated 6 times, giving in all 12 rats at each dose
level of unknown. Relative potencies were calcu-
lated on the basis of reduction in net granuloma
weight, with an empirical adjustment for gain or
loss of body weight of the animal during treat-
ment. One unknown showed a dose response curve
with slope significantly different from the re-
maining compounds, so that its potency was
indeterminate. Relative potencies of the remain-
ing steroids were: cortisol acetate = 1.00; 9achloro,
21 desoxy-cortisone = 2.38; 9achloro, 21 desoxy-
cortisol = 0.59; 9efluoro, 21 desoxy-cortisol =
0.34. The dose response curve for 9efluoro, 21 des-
oxy-cortisone crossed that of the standard,
where it would have a potency of 1.00, in the
neighborhood of a dose of 100 gamma. The com-
pound showing highest anti-inflammatory activ-
ity, Qachloro, 21 desoxy-cortisone, had _ the
disadvantage of a-correspondingly high salt
tetaining activity as determined in this laboratory
by C. M. Kagawa.

1423. Persistence of reserpine action after its
disappearance from brain. Sipney M. HEss,*
ParkuHurst A. SHORE* AND BERNARD B. BropieE.
Lab. of Chemical Pharmacology, Natl. Heart Inst.,
Natl. Insts. of Health, Bethesda, Md.

The hypothesis has been advanced that the
central actions of reserpine are mediated through
serotonin in brain. This is based on findings that:
1) reserpine and serotonin have central actions
in common including potentiation of hypnotics,

lysergic acid diethylamide, 3) reserpine changes
serotonin in brain from a bound to a free form,
4) only the pharmacologically active Rauwolfia
alkaloids effect the release of serotonin. The
present studies supply additional support for the
role of serotonin in reserpine action by demon-
strating that the change in serotonin and pharma-
cologic effects persist long after reserpine has
disappeared from the brain. Rabbits received
5 mg/kg of reserpine intravenously, and at various
times following administration brains were
analyzed fluorometrically for reserpine and
serotonin. Reserpine rapidly enters brain, the
maximal concentration (about 8 ug/gm) being
found 10-15 min. following intravenous injection.
Levels declined rapidly and reserpine could not be
detected after about 8 hr. (<0.05 ug/gm). How-
ever, the marked pharmacologic effects of reser-
pine, including sedation, persisted for about 48 hr.
Serotonin was almost completely released from
brain depots within 4 hr., remained at a constant
low level for about 30 hr., then rose slowly to
normal levels by the 7th day. From these data it
appears that reserpine per se does not cause its
central effects but rather by producing a per-
sistent change in brain serotonin.

1424. Chromatographic method for extracting
acetic acid from animal tissues and incuba-
tion fluids. Liz Tsinac Hiren anp Tuomas N.
BuRBRIDGE (introduced by Hamitton H.
ANDERSON). Depts. of Pharmacology and Expil.
Therapeutics, Univ. of California School of Medi-
cine, San Francisco, and Faculty of Medicine,
Univ. of Indonesia, Djakarta.

A study of the metabolism of ethanol requires

a satisfactory method for the extraction and

identification of acetic acid. Such a method, which

is applicable to biologic fluids and tissues, has
been developed for animals. A rat is killed and the
entire gastroenteric tract is removed immedi-
ately (to eliminate acids from intestinal flora).

The chest and cranial cavities are opened and the

animal is immersed in liquid air or nitrogen. The

frozen carcass is then pulverized manually and
then in a Waring Blendor with about 500 ml of

0.45% zine sulfate. Next, approximately 0.1 Nn

sodium hydroxide is added to pu 8, followed by

steam distillation to remove substances volatile in
alkaline solution. The residue is acidified to about
pH 2, and steam distillation continued to remove
volatile acids. The distillate is titrated with 0.1 Nn

NaOH to pu 9 to determine amount of acid present.

This distillate is then evaporated to dryness,

acidified with H:SO, and run through a silica

column coated with R—NH, (acid-base) indicator

(modified from method of RaMsEY AND PATTERSON,

1945). For fluids from Warburg flasks, the method

is identical; pulverization is unnecessary. Total

438

acetic acid recovery averages approximately 17.9
mg/200 gm rat carcass. Four other acids are
separated on the column, one of which is formic
acid. The other 3 have not yet been identified.
If a known mixture containing formic, acetic,
propionic and butyric acids is run through the
column, approximately a 95% recovery of each
acid occurs. (Supported, in part, by a grant from
the Giannini-Bank of America Fndn.)

1425. Blood esterase activity following par-
enteral trypsin and chymotrypsin. ROBERT
G. Hitt anp J. W. Bastian (introduced by N.
Ercout). Dept. of Pharmacology, Armour Labs.,
Chicago, Ill.

The existence of inhibitors for trypsin and
chymotrypsin in plasma is well established and it
is generally considered that the introduction of
tolerated amounts of these enzymes into the
blood results in nearly total loss of protease
activity. In contrast to this, we found that the
esterase activities of these enzymes were sig-
nificantly less inhibited in the presence of plasma.
Determinations using acetyl-l-tyrosine ethyl
ester as a substrate for chymotrypsin and p-tosyl-
l-arginine methyl] ester as a substrate for trypsin
showed that the free esterase activities of these
enzymes in the presence of rabbit plasma are
directly proportional to the total amount of
enzyme added. In the presence of 0.5 ml plasma,
with chymotrypsin 9% and with trypsin 35% of
the total esterolytie activity remained free. These
values were obtained following a 2-min. incuba-
tion between the enzyme and plasma, but other
experiments showed no detectable change in free
esterase activity even up to 18 min. incubation.
Esterase activity was easily measured in plasma
samples from rabbits treated intravenously with
3 mg/kg chymotrypsin or 1 mg/kg trypsin. No
detectable increase in circulating esterase activity
was observed in rabbits treated intramuscularly
with 10 mg/kg chymotrypsin or 7.5 mg/kg trypsin
in oil. This suggests that the anti-inflammatory
effect observed following intramuscular injections
is not mediated by the systemic absorption of
enzyme reSulting in free protease activity.

1426. Action of ryanodine on isolated kitten
auricle. IRA W. HiLtyarp AND LEONARD
Procrta (introduced by Erwin E. NELson).
Dept. of Pharmacology, St. Louis Univ. School of
Medicine, St. Louis, Mo.

Ryanodine, which produces a rigor of skeletal
muscle, produces an irreversible contractile failure
of isolated, spontaneously beating kitten auricles.
This occurs with concentrations of ryanodine as
low as 5 X 10-° m. The rate of depression of con-
tractility is curvilinear and dependent upon the
concentration of ryanodine employed. Complete
failure may occur within 3 min. with concentra-

FEDERATION PROCEEDINGS

Volume 16

tions of 10-4 m, and within 90-100 min. with the
lowest effective concentration mentioned above.
Although the contractile failure has proven to be
irreversible, there occasionally is some spon-
taneous recovery after several hours, but this is
never more than 40-50% of control activity.
Concentrations of 5 X 10-° M are ineffective but
tend to produce refractoriness in some prepara-
tions. While the rate of ryanodine failure is con-
centration dependent the degree of failure is not,
as complete inhibition of contractility occurs with
all effective concentrations. When ryanodine is
washed from the bath within a minute of its addi-
tion, contractile failure is not prevented. The rate
of failure is significantly reduced, however.
Ouabain, creatine phosphate and adenosinetri-
phosphate have not proven to be ryanodine
antagonists. Pretreatment with cysteine and
glutathione has also proven nonprotective.
Adrenaline, calcium and isopropylarterenol are
very effective in restoring the contractile capacity
of the failed auricle, but in experiments thus far
this antagonism has proved to be only temporary.
The spontaneous rate, although depressed, is
rarely decreased by more than 40% of control.
(Supported by a grant from St. Louis Heart
Assoc.)

1427. Effect of total spinal anesthesia upon
blood pressure responses to histamine,
James G. HILTon AND SHELBY C., ReErp.* Dept.
of Pharmacology, Univ. of Mississippi Med.
Ctr., Jackson.

Previously it has been shown that denervation
of the pressoreceptors does not significantly
affect the blood pressure responses to injected
histamine when the differences in the control
blood pressure due to denervation are considered.
It was the purpose of this investigation to deter-
mine the effects of removal of all reflexes at the
spinal level upon the blood pressure responses
to injected histamine. This study was carried out
by comparing the blood pressure responses to
graded doses of histamine in the dog before and
after total spinal anesthesia. The results of these
experiments showed that the minimum to which
the blood pressure would fall as the result of the
doses of histamine was lower but the actual fall in
blood pressure was less in animals under total
spinal anesthesia. The duration of the fall in blood
pressure was significantly longer at the higher
dosage levels of histamine following total spinal
anesthesia despite the fact that the actual fall in
blood pressure was less. By measurement of
the angle of fall in blood pressure and the angle of
return of the blood pressure to control levels these
differences in duration of blood pressure response
seem to be due to a slightly slower rate of fall and
to a much prolonged rate of recovery of the blood
pressure in animals under total spinal anesthesia.

th
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1429

ne 16

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March 1956

1428. Physostigmine transport in human
erythrocyte. W. C. HoLLANp Anp G. V. Aupi-
TORE.* Dept. of Pharmacology, Vanderbilt Univ.
School of Medicine, Nashville, Tenn.

The uptake of the physostigmine cation by
human erythrocytes in neutral or weakly acid
medium has been investigated. Within 1 min.,
the earliest observation, the reaction was 80-90%
complete. At the end of 120 min. the system had
reached equilibrium. Curves relating physo-
stigmine uptake as a function of physostigmine
concentration can be conveniently expressed as 2
straight lines, suggesting that physostigmine
is equilibrating with 2 distinct cellular com-
partments with different affinities for the cation.
The compartment with the greater affinity,
representing the cell surface and containing the
enzyme cholinesterase, is depicted by the line
with the steep slope. The distribution ratio for
physostigmine [(P)cen/(P) medium] between the sus-
pending medium and this phase has a mean value
of 7.8+0.3. The corresponding ratio for the other
compartment (probably the cytoplasm) is 1.56+
0.4. In the presence of high concentrations of
acetyl choline (10-*-10-? m) the rate of uptake
and the equilibrium distribution ratio of physo-
stigmine were slightly diminished. Calculations
from equilibrium data showed that the maximum
amount of the cation that was displaced from the
cell by these concentrations of acetyl choline was
of the order of 10-*-10-7 m of physostigmine per
liter of cells. It would appear from the analysis
that if the acetyl choline system plays a role in ion
transfer processes, it could affect the transport
rate of only asmall fraction of the total exchanging
cell cations (<1077 m/I.)

1429. Inhibition of poliomyelitis virus by
metabolic analogues in mammalian tissue
cultures. ARIEL C. HOLLINSHEAD* AND PAUL
K. Situ. Dept. of Pharmacology, George Wash-
ington Univ. School of Medicine, Washington,
Do:

Inhibition of type I poliomyelitis virus (WS
strain) in monkey testicular tissue cultures with
additional testing in monkey kidney and human
HeLa culture, by amino acid analogues, purine
and pyrimidine analogues, and certain sulfur
compounds have been studied. Among the active
compounds were 2-methyl-5-butyl-4,6-dihydroxy
pyrimidine, 5,6 dioxyuracil, 5-aminouracil,
|-phenyl-4-anilinopyrazolo (3,4-d) pyrimidine,
2-methylmercapto-ethanol, 5-oxide phenothiazine,
10-oxide phenoxathiin, 10,10-dioxide phenox-
athiin, phenyl sulfone, 3-thiophenecarboxylic
acid, 6-thianonaphtenamine, 3-thiophenealanine,
2-thiophenealanine, sulfonium compound-tris
(2-hydroxyethyl) chloride, 2-n-butyl-2-ethyl-1,3-
propanediol dicarbamate, isoguanine, carbanilic
acid-2-chloroethyl ester, d-leucyl-l-tyrosine,

EXUM

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

439

w-methylpantothenate, and 4-methoxy-6-nitro-
benzimidazole. The inhibitors have been studied
with regard to possible mechanisms of inhibition
by tests for direct viricidal action, for direct
effects on the tissue, for reversal by metabolites
and for inhibition of the growth of the virus.
There were some competitive inhibitions such as
reversal of inhibition of w-methyl-Ca-panto-
thenate by pantothenate, 5-aminouracil by cyto-
sine, and the thiophenealanines by phenylalanine.

1430. Effect of known metabolic antagonists
on body -temperature control of intact rats.
Dorsty E. Houitrxamp, Artuur E. Hemine,
Doris B. HuntsMan,* MarGaret C. Dogcert*
AND ALFRED R. Maass.* Research and Develop.
Div., Smith, Kline and French Labs., Philadelphia,
Pa.

Homeothermia of the rat starts at about 18
days of age (HiLL, R. M. Am..J. Physiol. 149: 650,
1947). The succinicdehydrogenase enzyme activity,
which is low at birth, reaches the adult level at
about 15 days of age (PortTER, SCHNEIDER AND
LieBu. Cancer Research 5: 21, 1945). We therefore
hypothesized that homeothermia might be de-
pendent in part on oxidative metabolism. To test
this, we utilized homeothermic intact rats (150-200
gm in weight) and injected them subcutaneously
with sublethal and relatively ‘nontoxic’ doses of
known oxidative antagonists. Body-temperature
measurements were made by rectal (high colonic)
thermometer before treatment and }, 1, 2 and 3 hr.
after treatment. Rats were kept at room tempera-
ture (25°C). Sodium malonate at 1.5, 2.0 and 3.0
gm/kg lowered the mean body temperature by
0.3 to 0.6°C. Potassium cyanide at 5, 6 and 8
mg/kg lowered it by 0.5 to 2.3°C. Sodium azide
at 10 and 25 mg/kg lowered it by 1.0 to 2.2°C.
Additional work is necessary to explain these
findings, but we feel that they may be quite
important in providing new leads for the elucida-
tion of 1) the heat-producing aspects of the
mechanism of normal body-temperature regula-
tion and 2) the mechanism of at least some patho-
logical fever states.

1431. Reserpine effect in medullary vaso-
motor mechanism of the cat. Bert 8S. Hor-
witz* anv S. C. Wana. Dept. of Physiology,
College of Physicians and Surgeons, Columbia
Univ., New York City.

Sixteen vagotomized cats weighing 3.0-4.1 kg
under Nembutal anesthesia were examined for
medullary effects of reserpine. Using the stereo-
taxic technique the medullary vasomotor region
was stimulated with bipolar concentric electrodes
and rectangular pulses (a range of low voltages,
1.5-3 v., and frequency of 50/sec.). The arterial
blood pressure was recorded from the femoral
artery. The pressor effects produced by medullary

440

stimulation varied from 12 to 1388 mm Hg with the
majority of responses between 50-80 mm Hg. In
the same series, occlusion of the carotid arteries
for 40 sec. in 10 of the cats produced increases of
blood pressure of 55-112 mm Hg with an average
increase of 80 mm Hg. Observation for 2 hr. after
i.v. reserpine administration (0.4-1.0 mg/kg in
glycol or citric acid solution) revealed a hypo-
tensive response of 0-60 mm Hg with an average
drop of 25 mm Hg. Responses to direct electrical
stimulation were markedly reduced and in many
cases completely obliterated, with a range after
reserpine of 0-80 mm Hg and most of the re-
sponses less than 50 mm Hg. Responses to occlu-
sion of the carotid arteries were reduced to 25-100
mm Hg with the average response at 49 mm Hg.
One cat prepared as above was injected with a
citric acid placebo which had no effect on blood
pressure or response to electrical stimuli or carotid
artery occlusion. Upon termination of the experi-
ments the brains of the animals were examined
grossly to confirm the location of the electrode
tip. A series of experiments on cats to determine
the effects of reserpine on responses from direct
electrical stimulation of the posterior hypo-
thalamus is now in progress.

1432. Acute tolerance to mescaline in the dog.
MicuakE. J. Hosko, Jr.* anp RicHarp TIsLow.
Wyeth Inst. for Med. Research, Radnor, Pa.

With the increased use of mescaline as an
experimental tool in psychotherapy, it was deemed
of importance to determine effects of repeated
mescaline administration on the behavior pattern
of unanesthetized dogs, and on blood pressure
responses of pentobarbitalized animals. Repeated
intravenous injections of mescaline sulfate (5
mg/kg) produced decreasing carotid blood pres-
sure depressions. Mean blood pressure responses to
mescaline injected at / hr. intervals were 60, 47,
30 and 12% of the initial level. Dogs pretreated
with mescaline on the day preceding the blood
pressure test showed, upon initial mescaline
challenge, a mean blood pressure depression of
30%, subsequent mescaline injections at $hr.
intervals elicited decreasing responses of 20 and
12%. Isbell et al. (Federation Proc. 14: 354, 1955)
described tolerance to mental effects of another
hallucinogenic agent, diethylamide of lysergic
acid (LSD-25) in man. We tested the possibility
of cross-tolerance between mescaline and LSD-25
in anesthetized dogs pretreated with LSD-25; no
modification of the typical mescaline blood pres-
sure fall was observed. The distinctive behavior
pattern produced by an initial mescaline challenge
(20 mg/kg, i.v.) in the unanesthetized dog was
modified when subsequent challenges were given
within 24 hr. The over-all intensity of the follow-
ing responses was diminished: catatonia, passive
negativism, arched back and head hang, ap-

FEDERATION PROCEEDINGS

Volume 16

parent anxiety, eyewiping and clawing at mouth,
Symptoms not decreased in intensity were my-
driasis, labored respiration, compulsive head
shape and facial wrinkling. Essentially, no toler-
ance was observed if the second mescaline chal-
lenge was given 72 hr. or later.

1433. Concentrations of free morphine in
brain after pretreatment with SKF 525A.
Erxicut Hosoya (introduced by Yosuito
Kopayasut). Dept. of Pharmacology, Keiogijuku
Univ. Med. School, Tokyo, Japan.

It has been generally recognized that SKF
525A (8-diethylamino diphenylene propyl acetate)
potentiates the analgesic action of morphine in
rats. Several explanations have appeared for the
mechanism of this potentiation (Brop1e et al.,
KENSLER, Bropy aND Hosoya). Woods finds that
only free morphine has analgesic action and bound
morphine has no such action. Therefore, we
determined the concentration of free morphine
in brain after morphine with or without pretreat-
ment with SKF 525A. White male rats weighing
150 gm were used. SKF 525A 100 mg/kg. was ad-
ministered orally to 10 rats 40 min. after morphine
injection. Morphine HCl was injected intra-
peritoneally. The rats were decapitated 30 min.
after morphine injection and the whole brain
including cerebellum was analyzed. Free morphine
in brain was determined by the method of Woods
et al. (J. Pharmacol. & Exper. Therap. 111: 64,
1954). The mean concentration of free morphine/
1.0 gm brain tissue (10 rats) after i.p. injection of
4 mg/kg morphine without pretreatment was
5.66+2.0 y (as free base). The mean concentration
of free morphine after i.p. injection of 4 mg/kg
morphine with pretreatment of SKF 525A (10
rats) was 5.70+2.3 y. The concentration of free
morphine after i.p. injection of 150 mg/kg mor-
phine without pretreatment was 10.02+1.9 y (6
rats). The concentration of free morphine after
i.p. injection of 150 mg/kg morphine with pre-
treatment of SKF 525A was 11.3+1.9 y (6 rats).
Since no significant difference occurs in the con-
centration of free morphine in brain between
SKF 525A pretreated and nontreated rats we con-
clude that the potentiation of morphine analgesia
by SKF 525A does not depend upon the change
of the distribution of free morphine in brain.

1434. Clinical studies of morphine-nalorphine
combinations. R. W. HoupE anp S. L. WaL-
LENSTEIN.* Memorial Ctr., New York City.

A double blind, controlled study of the analgesic
effectiveness of 10 mg of morphine sulfate com-
bined with 1 mg of nalorphine hydrochloride in 14
hospitalized cancer patients showed the mixture
to be significantly superior to a sterile saline con-
trol and, except for the lst hr., about as effective
as 10 mg of morphine alone. Further studies were

me 16

.0uth,
e my-
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toler-
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525A.
SHITO
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SKF
etate)
ine in
or the
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brain
rphine
Woods
11: 64,
phine/
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change
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. WAL-

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nixture
ne con-
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March 1956

then carried out on morphine-nalorphine mixtures
containing 5 or 10 mg of morphine in which the
proportions of morphine to nalorphine were 8:1,
4:1, 2:1 and 1:1. Corresponding 5 or 10 mg doses
of morphine sulfate alone, and a sterile saline
placebo, were included as standards and control,
respectively. Cross-over comparisons were carried
out in 38 patients. Pain relief was estimated on the
basis of patients’ hourly reports of changes in
pain intensity for a 6-hr. period after intra-
muscular drug administration. A biphasic slope
of analgesic effectiveness of these combinations
was obtained. This was interpreted as indicating
that nalorphine not only progressively interferes
with the analgesic effect of morphine but also
exerts its own alagesic effect as the amount of
nalorphine in the mixture is increased. It was also
noted that the incidence of volunteered and
observable side effects increased in direct propor-
tion to the amount of nalorphine in the mixture
and in a separate limited study of the respiratory
effects of the mixtures in normal volunteers that
the combinations produced as much or more
respiratory depression than morphine alone.

1435. Amebacidal properties of N,N!-di-
piperonyl-5,ll-diaminopentadecane dihy-
drochloride. ARSENY K. HRENoFF* AND
Hamitton H. Anprerson. Dept. of Pharma-
cology and Exptl. Therapeutics, Univ. of Cali-
fornia, San Francisco.

Seven derivatives of diaminodecanes were
tested for possible action against E. histolytica
in vivo and in vivo. They were water soluble at
pH 4-5 and, being weak bases, were subject to
hydrolysis and precipitation at higher px values.
In vitro, in comparison with emetine hydrochloride
(active in the range of 1:100,000 at px 7.0), one of
the decanes tested proved to be more than twice
as active as emetine. This was N , N!-dipiperonyl-
5,11-diaminopentadecane dihydrochloride at
1:256,000 dilution against F-22 strain of E. histo-
lytica in association with a streptobacillus during
48 hr. exposure at 37°C. In macaques, naturally
infected with EZ. histolytica, daily oral doses of 25,
50 and 75 mg/kg were given over 10 days to 6
animals. Five of the 6 remained clear during a
follow-up period of 2-3 months. One animal
given 25 mg/kg recurred 2 wk. after therapy. One
other (among 5 cleared) developed tuberculosis
and died at the end of follow-up. Loss of weight
of approximately 10% was noted in 3 animals at
higher dosages, and in 2 animals, 5-8% loss was
noted at lower dosages. Diarrhea occurred during
the first 5 days of therapy. (Supported, in part,
by the Natl. Insts. of Health, PHS, and the
Miles-Ames Labs., Elkhart, Ind.)

1436. Renal excretion of some iodinated amino
acid derivatives by the dog. K. C. Huana*

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

441

AND P. K. Knoerex. Dept. of Pharmacology,

Univ. of Louisville, Louisville, Ky.

It has been shown by many investigators that
amino acids are extensively reabsorbed by the
renal tubules. In the last few years some iodinated
amino acid derivatives have been studied in this
laboratory for their behavior in renal excretion,
toxicity, protein binding and their opaque proper-
ties in kidney visualization. Dogs were anesthe-
thized with sodium amytal, and creatinine clear-
ance was used to measure the glomerular filtration
rate. The iodinated amino acid compound was
dissolved in an equimolar alkaline solution and
was injected intravenously in priming doses and
by continuous infusion. It was found that di-
iodotyrosine, its N-benzoyl and N-carbethoxy
derivatives, N-2-iodophenyl-a-alanine and N-2,4-
diiodophenyl-8-alanine, had a negative T value;
they were reabsorbed by the renal tubules. The
2 acetylated, N-acetyl and diacetyl, and N-(car-
boxymethyliminocarbonyl) derivatives of diiodo-
tyrosine and N-(2,3,5-triiodobenzoyl) tyrosine
had a higher clearance than the creatinine clear-
ance; they were excreted by the renal tubules.
All these amino acid derivatives were partially
bound with plasma protein, ranging from 30 to
90%. When diiodotyrosine was given in a total
dose of 1.8 gm/kg, a nephrogram was shown and
the renal cortex contained a large quantity of
iodine. There was no nephrogram produced with
the other compounds, because their given dose
was low, either due to shortage of the material or
due to their high toxicity.

1437. Comparison of osmotic effect of sodium
sulfate and cyclamate (cyclohexylsulfa-
mate) salts in alimentary tract of the rat.
Kao Hwang, R. Wiseman,* D. T. AsHER* AND
R. U. Rosinson.* Abbott Labs., North Chicago,
Til.

Cyclamate salts and sodium sulfate were
studied in unstarved rats regarding their relative
laxative potency after single oral doses. The
percentage of animals showing positive response
within 8 hr. was determined. The apparent oral
EDw’s in gm/kg for sodium cyclamate, calcium
cyclamate and sodium sulfate were, respectively,
1.9, 2.8, and 1.6 (about 30-40 times the possible
human intake per day for the cyclamates). How-
ever, the actual EDw’s in mOs/kg after being
corrected for both the amount absorbed during
the test and the apparent degree of ionization
were 15.1, 15.2 and 14.8, respectively. Motility of
the alimentary tract was measured simultaneously
by the passage of carbon suspended in the solu-
tions of the salts tested. No significant difference
among these salts was observed in these studies.
The mechanism of the laxative action of the
sodium salts was also explored. Single oral doses
of the S*-tagged salt were given to starved

442

rats. The free fluid present in the alimentary tract
was measured at the end of 13 hr. and the salt
concentration was analyzed. There was a high
degree of correlation between the corrected
osmotic dose and the volume of the fluid retained
for either sodium:sulfate or sodium cyclamate. No
significant difference was found between the
slopes and intercepts for both salts on covariance
analysis.

1438. Individual differences in response to
drugs. S. Irwin, M. SLaBoxk* anp G. THomas.*
Research Div., Schering Corp., Bloomfield, N.J.
A highly significant correlation was found

between the spontaneous motor activity level of
the individual male Carworth rat and its response
to psychomotor stimulants (pipradrol, metam-
phetamine) and psychomotor depressants (chlor-
promazine, pentobarbital, morphine). Two-hour
control activity measurements in Wahmann ac-
tivity drums were obtained prior to drug or saline
treatment, and the individual initial measure-
ments correlated with a 3-hr. measurement of
activity following treatment. The square root
transformation was used to secure additivity and
constant variance. The concomitant variate
was relatively stable in the same animal over a
2-month period. On the square root scale, saline
and stimulating drugs gave a family of parallel
lines when 3-hour activity was plotted against
control activity. The lines exhibited the same
slope but different intercepts. The depressant
drugs gave a family of nonparallel lines with
slopes approaching zero. In terms of the arith-
metic number of revolutions, the stimulants and
depressants produced a considerably greater
effect in animals with high control activity than in
animals with low spontaneous activity. The
control activity levels of the individual animals
were poorly correlated with their affective state
(fearfulness, placidity, aggressiveness), body
weight, B.M.R., latency for the tonic extensor
component of M.E.S. and response to a variety
of other drug actions. The observed correlation,
therefore, would appear to be on a selective
basis and unrelated to the general physiological
status of the animals.

1439. Attempted addiction to nalorphine.
Harris Ispetu. Natl. Inst. of Mental Health,
Addiction Research Ctr., PHS Hosp., Lexington,
Ky.

In both nontolerant former addicts and non-
addicts, nalorphine subcutaneously in doses of
5-10 mg (nonaddicts) or 10-30 mg (former addicts)
caused subjective sensations of dizziness, light-
headedness, tremors, warmth, difficulty in speech,
garrulousness, uninhibited behavior, nausea and,
in some cases, vomiting. Such effects disappeared
in less than 1 hr. and were succeeded by drowsiness

FEDERATION PROCEEDINGS

Volume 16

and sleep. Former addicts disliked the drug.
With the higher doses confusion and visual
hallucinations occurred in some former addicts.
Objectively, nalorphine reduced body tempera-
ture, depressed respiratory minute volume and
caused slight pupillary constriction. Six former
addicts were given 10 mg of nalorphine every 6 hr.,
increasing in 14 days to 25-35 mg every 6 hr. Two
patients withdrew from the experiment because
of hallucinations. The other 4 continued but
would not permit elevation of the dose above
100-130 mg daily. Hallucinations disappeared after
2 wk. No definite symptoms were observed after
abrupt withdrawal of nalorphine after 28 days
addiction. Four other addicts received nalorphine
in doses increasing to 7-12 mg every 4 hr. for 25-31
days and 1 patient received nalorphine in doses
increasing to 9 mg every 3 hr. for 42 days. Dis-
agreeable side effects were less with such small
doses more frequently administered but again
patients disliked medication. No definite symp-
toms of abstinence occurred following withdrawal
in these last 5 men. Addiction liability of nalor-
phine is low or nonexistent.

1440. Effect of chlorpromazine, reserpine and
‘Frenquel’ on LSD reaction. Harris ISBELL.
Natl. Inst. of Mental Health, Addiction Research
Ctr., PHS Hosp., Lexington, Ky.

It was hoped that blocking or reversal of psy-
chosis induced by the diethylamide of lysergic
acid (LSD-25) could be used as a screen for pre-
dicting clinical usefulness of new tranquilizing
agents. Intensity of LSD reaction was evaluated
by a questionnaire and a short mental status
examination supplemented by measurements of
pupillary size, knee jerks and blood pressure. All
experiments were ‘double-blind’ and included in
the same patients combinations in random order
of LSD-placebo-tranquilizer-placebo, LSD-tran-
quilizer-placebo, LSD placebo-tranquilizer and
LSD-tranquilizer. In 5 experiments using 39
patients in which 50-100 mg of chlorpromazine
were given 30 min. prior to 40-60 ug LSD, a strong,
but not always significant, trend to reduction in
the intensity of the reaction was observed.
Seventy-five mg of chlorpromazine orally 1} hr.
after 60 ug LSD did not reduce the intensity of the
LSD reaction, whereas 25 mg i.m. did reduce its
intensity. One mg of reserpine orally 10 and 2 hr.
before or 2.5 mg orally 22, 10 and 2 hr. before
60 wg LSD did not reduce the intensity of the
LSD response. Two mg of reserpine intramus-
cularly 22, 10 and 2 hr. before LSD intensified
the LSD reaction. Twenty mg of 4-piperidyl-
diphenyl-carbinol (Frenquel) t.i.d. for 7 days
prior to 60 wg LSD had no effect. Sixty mg of
Frenquel i.v. and 40 mg i.v. 2 and 3 hr. after
100-200 wg LSD also failed to influence the reac-
tion. Because of the results with reserpine, the

So = = @ = woe lee ee

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drug.
visual
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pera-
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ormer
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. Two
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. Dis-
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March 1956

LSD reaction is not an effective screen for de-
tecting tranquilizers.

1441. Effect of chlorin derivative on perfused
hypodynamic frog heart (Motion picture).
L. H. Joprey, M. J. HENDRICKSON AND R. R.
BervueErry (introduced by A. R. McIntyre).
Univ. of Nebraska College of Medicine, Omaha.
The film shows the perfused frog heart arranged

for kymographic and ECG recording. The normal

heart activity is then rendered hypodynamic by
the withdrawal of calcium from the perfusion
fluid. The addition of approximately 1 ppm of
chlorin derivative is shown to restore the heart
activity to normal and ‘supernormal’ activity.

This action of chlorin derivative resembles the

action of cardiac glycosides.

1442. Central actions of a convulsant barbi-
turate. EuGENE R. Jotuy* anp Epwarp F.,
Domino. Dept. of Pharmacology, Univ. of Mich-
igan, Ann Arbor.

The effects of sodium 5-ethy],5(1,3-dimethyl-
butyl) barbiturate were determined in rabbits
in which the basilar artery was ligated at the mid-
pontine level. Intravertebral injections of 0.5-3.0
mg/kg of convulsant barbiturate produced marked
motor activity accompanied by EEG arousal—
similar to that produced by intravertebral injec-
tions of pentylenetetrazol or picrotoxin. After
momentary depression, respiratory and vasomotor
activity were markedly stimulated. By internal
carotid injection slightly larger doses (1.5-3.0
mg/kg) of convulsant barbiturate produced com-
parable overt effects. Continuous spike-like dis-
charges or ‘persistent spindling’ patterns were
observed in the EEG. With large or repeated doses
EEG depression occurred. Stimulant effects of
convulsant barbiturate were much less prominent
in anesthetized animals. Respiratory depression
produced by thiopental given intravertebrally was
potentiated by convulsant barbiturate. Respira-
tory arrest could also be produced if large or
repeated doses of the drug were injected intra-
vertebrally. Microscopic examination of serial
sections after intravertebral dye injections re-
vealed dye penetration a few millimeters rostral
to the level of ligation. Dye extended caudal to
thoracic spinal cord. The effects of intravenous
convulsant barbiturate on bulboreticular facilita-
tion and inhibition of the patellar reflex of cats
given chloralosane depend upon the depth of
anesthesia. With light anesthesia 1 mg/kg of
convulsant barbiturate produced enhancement of
the patellar reflex as well as an increase in facilita-
tion and inhibition. In deeply anesthetized prepa-
rations the patellar reflex was markedly depressed
after initial enhancement. Bulbar facilitatory
effects persisted to a diminished degree. It is con-
cluded that this agent has independent stimulant

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

443

and depressant actions on several levels of the
central nervous system.

1443. Biphasic response in sodium excretion
following 11,17-oxysteroid administration
in adrenalectomized rat. C. M. KaGgawa*
AND C. G. Van ArMAN. Div. of Biological Re-
search, G. D. Searle and Co., Chicago, Til.

The acute actions of 11,17-oxysteroids on
sodium excretion in adrenalectomized rats are
ill defined. Small doses have generally increased
output, and relatively large doses have shown no
effect or a slight reduction, suggesting that the
activity of these steroids may be influenced by a
time factor. Results from studies in adrenalec-
tomized rats indicate that 0.5-1.0 mg cortisone
and hydrocortisone, administered as a single
dose, cause an initial retention followed by
a strong sodium diuresis, with the total net effect
clearly sodium loss. These charges in sodium
excretion are biphasic in the presence of con-
tinued potassium and water losses. Progressively
smaller doses quantitatively reduce the magni-
tude and duration of the biphasic phenomenon.
Using a single collection of urine, it follows that
various segments of this biphasic response will be
measured depending on the dose of the steroid.
Retention may seem to be the predominant phe-
nomenon if the urine specimen measures the
primary phase. These data show that sodium
measurements may be strongly influenced by
biphasic responses. A weak sodium retaining
action, qualitatively like that in man, can be
demonstrated by proper sampling with 11,17-
oxysteroids in adrenalectomized rats.

1444. Effects of certain ribosides and ribotides
on cation transfer in human erythrocytes.
J.B. Kaun, Jr., Dept. of Pharmacology, Univ. of
Cincinnati College of Medicine, Cincinnati, Ohio.
Adenosine increased Na and K transport across

incubated cold-stored erythrocytes treated as

previously described (J. Pharmacol. & Exper.

Therap. 115: 305, 1955). The increase reached a

maximum of 50% higher than controls at 5 mm;

higher concentrations produced progressively less

augmentation. AMP caused augmentation of
transport reaching a maximum of 20% above
controls at 2.5 mM; higher concentrations pro-

duced less increase, and concentrations of 10 mm

and above decreased cation transfer relative to

controls. ADP and ATP were purely inhibitory,

0.625 mm inhibiting by 5%, and 40 mm inhibiting

completely. None of the above compounds affected

the block of transport induced by cardiac glyco-
sides. Cytidine and cytidylic acid were tested in
red cells under the same conditions and had
purely inhibitory effects. Concentrations of
cytidine higher than 5 mo inhibited transport, as
did cytidylic acid at 2.5 mm and higher. The latter

444

inhibited transport completely above 20 mm.
The hypothesis is made that adenosine has two
effects on transport, one to increase it, probably
dependent upon the role of adenosine in energy
metabolism inside the red cell, and one to decrease
it, depending upon some other effect of adenosine
inside or outside the cell. The adenosine phos-
phates, which probably do not enter the cell, have
only the inhibitory effects, with the exception of
low concentrations of AMP, which may enter the
cell. Cytidine and cytidylic acid, having no role
in energy metabolism, have only the inhibitory
effects. Effects of other ribosides and ribotides
will be reported. (Public Health Service Grant
H-1506 (C).)

1445. Qualitative variation of serum cho-
linesterase activity in man as defined by
dibucaine-numbers. W. Katow, K. GENEst*
AND N. Sraron.* Dept. of Pharmacology, Univ.
of Toronto, Toronto, Canada.

The simplest test found so far, which is suitable
for the screening of human sera for qualitative
differences of serum cholinesterase activity, con-
sists in measuring the degree of esterase inhibition
by a given concentration of dibucaine. We observe
hydrolysis of 5 X 10-7 m benzoylcholine by serum
diluted 1:100 with phosphate buffer pu 7.4 at
room temperature in the spectrophotometer at
240 » (Canad. J. Biochem. & Physiol. 33: 568,
1955). The addition of 10-5 m dibucaine causes
usually 81 (S.D. +3.5) % inhibition of the esterase
activity. In rare cases the inhibition is only 10-
20%; intermediate degrees occur more often.
Hence we describe esterase characteristics by a
‘dibucaine-number,’ which is not directly related
to the esterase level in conventional units. The
dibucaine-number of most sera is around 80. A
lower dibucaine-number implies the following
differences from the usual picture: the Michaelis
constants K,, for all investigated substrates and
inhibitors are elevated. Relative reaction rates
for various substrates are altered. A change of
pH from 5.5-10 exerts only a weak influence on
Kn and reaction velocities with benzoylcholine.
Not influenced are the electrophoretic motility of
the enzyme (O. Smirures), the inactivation tem-
perature and the energy of activation. The di-
bucaine-number appears to depend on genetic
factors. (Aided by a Mental Health Grant from
the Ontario Dept. of Health.)

1446. Influence of potassium on renal tubular
excretion of basic organic compounds. A.
KANDEL (introduced by E. Burpine). Dept. of
Pharmacology, Tulane Univ. School of Medicine,
New Orleans, La.

Experiments designed to determine the in-
fluence of potassium loading on the renal tubular
excretion of basic organic compounds were per-

FEDERATION PROCEEDINGS

Volume 16

formed on trained, unanesthetized dogs. Glo-
merular filtration rate and renal plasma flow were
determined by usual methods. Potassium and
sodium determinations were made with an internal
standard flame photometer. The renal tubular
excretion of N’-methylnicotinamide (NMN) and
5 - methyl - 4 - phenyl - 1 - (1 - piperidyl) - 3 -
hexanol methobromide (Darstine) were compared
before and after the intravenous infusion of 0.5
mm KCl/min. Duplicate experiments were per-
formed on each animal after a period of forced
oral feeding with 10 gm KCl daily for 1 wk. Simul-
taneous infusion of KCl resulted regularly in a
reduction of the renal tubular excretion of Dar-
stine. Under similar conditions the renal tubular
excretion of NMN was unaffected. After the
period of forced feeding of KCl simultaneous
infusion of KCl produced a reduction in the renal
tubular excretion of NMN, as well as that of
Darstine. (Supported by a grant from the
Lousiana Heart Assoc.)

1447. Effect of previous morphine administra-
tion on subjective effects of nalorphine.
Artuur 8. Keats AND JANE TELFoRD.* Dept.
of Anesthesiology, Baylor Univ. College of Med-
icine and Jefferson Davis Hosp., Houston, Texas.
In a previous study (Knats aND MITHOEFER.

Federation Proc. 14: 356, 1955) it was shown that

nalorphine did not antagonize the respiratory de-

pression of morphine in man under all circum-
stances. Some or all of the factors of total dose of
morphine, frequency of morphine administration,
and time interval between morphine and nalor-
phine affected the antagonistic action of nalor-
phine. The present study was designed to deter-
mine if these factors also operate in determining
the subjective effects produced by nalorphine in
man. The effects produced by a single dose of

10 mg of nalorphine intramuscularly were com-

pared in 2 groups of postoperative patients.

One group received only a single preoperative

dose of narcotic and was given nalorphine more

than 24 hr. later. The second group received 40

mg or more of morphine in at least 4 doses over

20 hr. and was given nalorphine within 10 hr. of

the last morphine dose.

1448. Phosphate response in analgesic medi-
cation to rabbits. E. F. Kerra,* J. We1sBerc*
AND B. DEBorr. Dept. of Physiology and Phar-
macology, Univ. of North Dakota, Grand Forks.
Few observations have been made in regard to

phosphate reactions in narcotic administration.

Because of this, and in that it would be possible to

modify our glucose techniques so that observa-

tion on phosphate levels could readily be made on
the same samples, we decided to observe the
phosphate reactions in a series of animals. EKight-
een animals were submitted to medication once

ro ate nt 6S hlUC OR let OUelCUeklUlkelUC lel ee sll

ume 16

. Glo-
w were
n and
ternal
ubular
v) and
)-3-
npared
of 0.5
e per-
forced
Simul-
y ina
f Dar-
ubular
er the
aneous
e renal
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m the

listra-
phine.
‘ Dept.
f Med-
Texas.
OEFER.
nm that
ory de-
ircum-
dose of
ration,
nalor-
nalor-
. deter-
mining
ine in
lose of
> com-
itients.
erative
e more
ved 40
28 Over
hr. of

medi-
SBERG*
1 Phar-
Forks.
gard to
‘ration.
sible to
bserva-
nade on
‘ve the
_ EKight-
m once

March 1956

a week -with morphine (5 mg/kg), Nisentil (5
mg/kg), Levorphan (2.5 mg/kg) and saline (0.5
ml/kg) and drug administration rotated until a
latin square distribution was obtained. The
previously reported glucose response occurred
with a significant hyperglycemia at the reported
3 and 1 hr. observation. Again the peak hyper-
glycemia response was observed at the 1 hr. period
of observation. A phosphatemia was observed to
occur in significant levels at 4, 1 and 3 hr. obser-
vations. The minimum significant increase at the
} hr. period was 11.4% above comparable control
observation. The peak phosphatemia occurred in
the 4 hr. sample and remained significantly above
the saline control levels at 1 and 3 hr. The mini-
mum phosphatemia at the $ hr. observation was due
to morphine. Phosphate levels at this period were
for morphine 9.64 mg%, Nisentil 10.283 mg% and
Levorphan 9.83 mg% against a saline level of
8.43 mg%. Blood phosphates were below the pre-
medication control at the 6 hr. observation. The
premedication control level was 7.6 mg%. (Aided
in part by Natl. Insts. of Health Grant B645-
B645S.)

1449. Separation of formed elements of blood
with Cohn Blood Fractionator. KritH H.
Ketiy,* Howarp R. Brerman, Fauno L.
CorpEs* anp Harriet Watson.* City of Hope
Med. Center, Duarte, Calif.

The Cohn blood fractionator has permitted the
rapid separation of formed elements of fresh blood
under sterile conditions. Platelets, leucocytes and
erythrocytes may be collected separately depend-
ing on the conditions of fractionation. At speeds of
3800 rpm or less, platelet rich plasma is produced;
at 5000 rpm, at room temperature, a platelet and
white cell rich buffy coat with a hematocrit of
20% can be produced within 6-8 min. after dona-
tion of the blood. The blood and the fractions
must be collected in siliconized or plastic con-
tainers to avoid loss of platelets and white cells.
Blood from patients with anemia, polycythemia
or leukemia may be separated in the same fashion
as blood from normal donors, if an easily carried
out adjustment is made for the variation of packed
cell volumes. The results of fractionation of over
250u of normal fresh blood, together with the
results of fractionation of anemic, polycythemic
and leukemic blood will be presented in tabular
form. Blood fractions obtained in this manner
have been sterile and available for reintroduction.
Twenty-four patients, 8 adults and 16 children,
have received repeated infusions of such blood
fractions without untoward effect. Specific frac-
tions of buffy coat spun at rates between 3000 and
5000 rpm have produced a potent antihemorrhagic
component which has been effective in controlling
bleeding in the leukemias. Careful handling,

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

445

temperature control and storage conditions of
such fractions are essential toward maintaining
the viability of the formed elements.

1450. Study of meralluride excretion products
by chromatographic separation in patients
requiring mercurial diuresis. Bartis KENT,*
CaRROLL HANDLEY, JOHN STRAWN* AND JOHN
Moyer. Depts. of Pharmacology and Medicine,
Baylor Univ. College of Medicine, Houston,
Texas.

Recent studies have shown that meralluride is
normally excreted in the urine primarily as an
organic compound rather than inorganic mercury.
This study endeavors to determine if meralluride
is excreted in this same manner by cardiac pa
tients requiring mercurial diuresis. Two groups of
adult patients with proven heart failure without
significant renal impairment or electrolyte im-
balance were selected. One group had responded
with adequate diuresis to meralluride injections
at frequent intervals and the other group, con-
sidered mercurial resistant, had required hos-
pitalization for congestive failure. Urine controls
were collected before each study and 78 mg of
meralluride was injected either intravenously,
intramuscularly or subcutaneously. The next
24-hr. urine specimen separated into 6-hr. frac-
tionals was then evaluated by passing a measured
sample of each through a chromatography column
containing 5 gm of acid aluminum oxide. This
acted as a specific adsorbent for carboxylic acids
which includes meralluride and allowed other
mercury compounds including inorganic mercury
to pass through. The meralluride was then eluted
with 5% sodium carbonate and the mercury con-
tent of each fraction determined. The results
showed that 80-100% of the mercury was excreted
in 24 hr. by all routes of injection. The majority
of this was excreted in 12 hr. following intravenous
injection and in 18 hr. following both intra-
muscular and subcutaneous injection. In both
groups up to 98% was excreted in the carboxylated
form. That amount excreted in degraded forms
represented smaller quantities than that required
for diuresis with any known mercurial compound.
This study shows that there appears to be no
significant difference in the rate and form of ex-
cretion of meralluride by cardiac patients either
responsive or refractory to mercurial diuresis.

1451. Effect of Antabuse on action of par-
aldehyde in mice and dogs. M. L. KEPLINGER*
AND J. A. WELLS. Dept. of Pharmacology, North-
western Univ. Med. School, Chicago, Til.

It is presumed that paraldehyde is depoly-
merized in the body to acetaldehyde and that
acetaldehyde is then oxidized by aldehyde de-
hydrogenase. If the depolymerization is a re-
versible process, then interference with the

446

oxidation of acetaldehyde should delay the
disappearance of paraldehyde from the tissues
and, in turn, prolong its action. Tetraethyl-
thiuram disulfide (Antabuse) is known to inhibit
aldehyde dehydrogenase, and the present experi-
ments are concerned with the influence of this
substance on the actions and persistence of
paraldehyde. Antabuse administration (50-400
mg/kg i.p. and 200-800 mg/kg orally to mice and
100 mg/kg orally for 3 days to dogs) significantly
prolongs the sleeping time following paraldehyde
administration (120% in mice and 300% in dogs).
The toxicity of paraldehyde (LD in mice) was
not increased by Antabuse. The blood levels of
paraldehyde were slightly higher and much more
prolonged in Antabuse treated than in normal
dogs. Since the ‘awaking’ blood levels of par-
aldehyde were not different in the treated animals
from those of the controls, it is presumed that
prolonged sleeping time is simply a reflection of
prolonged paraldehyde persistence. Blood acet-
aldehyde levels are somewhat higher and more
prolonged in the Antabuse treated dogs receiving
paraldehyde than in the paraldehyde control
animals. However, the acetaldehyde levels are far
short of those achieved following the administra-
tion of ethyl alcohol to Antabuse-treated animals.
This is taken as additional support for the notion
that the paraldehyde to acetaldehyde reaction is a
reversible process.

1452. Cardiac glycosides in tumbling shock.
ALEXANDER C. Kryi* anp WIiLiiAM C. Norrtu.

Dept. of Pharmacology, Northwestern Univ.
Med. School, Chicago, Ill.
Szent-Gyorgyi observed that DCA, proges-

terone and certain cardiac glycosides were capable
of eliminating the ‘staircase’ phenomenon in the
isolated frog heart. He postulated that this might
be due to regulation of cell membrane permeability
to maintain favorable electrolyte balance. (Chem-
ical Physiology of Body and Heart Muscle, New
York: Acad. Press, 1953). This suggested trial of
cardiac glycosides prophylactically in traumatic
shock, a condition characterized by unfavorable
shifts in’ electrolyte. Accordingly, digitoxin
(0.625-5.0 mg/kg), ouabain (12.5 mg/kg), and
k-strophanthin (5.0 mg/kg) were administered
intraperitoneally to male Sprague-Dawley rats
22 hr. before tumbling in the Noble-Collip drum.
These glycosides exerted a protective effect equal
to or greater than atropine or Dibenzyline, the
most effective agents studied in this laboratory to
date. Preliminary attempts to implicate DCA
type activity as the mechanism of this protective
action have been inconclusive.
1453. Coronary vasodilator action of cin-
namyl Vonedrine (N-methyl-N-cinnamyl-

2-phenylpropylamine). Greratp A. KreEn,*

FEDERATION PROCEEDINGS

Volume 16

ALLEN W. GomoLL,* AND THEODORE R. SHER-
rop. Dept. of Pharmacology, Univ. of Illinois
College of Medicine, Chicago.

Cinnamyl Vonedrine (N-methyl-N-cinnamyl-
2-phenyl-propylamine) is an unusual coronary
vasodilator in that it produces an increased
coronary blood flow in the absence of any sig-
nificant changes in the blood pressure or heart
rate. These studies were performed in the opened
chest dog, anesthetized with pentobarbital sodium
and on artificial respiration with air. The cor-
onary blood flow was determined both by means
of the coronary sinus outflow technique according
to the method of Sherrod and by means of coronary
artery inflow, with a Shipley rotameter inter-
posed between a carotid artery and the circumflex
branch of the left coronary artery. Intravenous
doses of the drug ranging from 0.5-5.0 mg/kg of
body weight resulted in a maximal increase in
coronary sinus outflow of 75%, lasting from 10-
45 min. There was a transient hypotensive action
lasting for 20-45 sec. followed by a slight rise in
blood pressure of about 10 mm Hg with no sig-
nificant changes in heart rate. The drug was also
administered into the circumflex branch of the
left coronary artery in doses ranging from 100-
400 wg total. This produced an increased coronary
artery inflow of 10-73%, depending upon the dose.
Cinnamy!] Vonedrine effectively counteracted the
vasoconstrictor actions of Pitressin. The changes
in coronary artery inflow occurred in the absence
of any significant changes in either blood pressure
or heart rate. These results were compared with
those of papaverine. (Supported in part through
a research grant from the Dept. of Health, Edu-

cation and Welfare, Public Health Service.)

1454. Effect of reserpine and chlorpromazine
on positive reinforcement behavior in rats.
K. F. Kiuiam, J. Otps anp P. Bacn y Rita
(introduced by E. K. Kiiuam). Depts. of Physi-
ological Chemistry and Physiology, School of
Medicine, Univ. of Calif. at Los Angeles, Los
Angeles.

Reserpine and chlorpromazine were tested in
rats for their effect on reward by electrical stimu-
lation of the brain. Chronic electrodes were im-
planted in the amygdala, septum or hypothala-
mus and the animals were trained to stimulate
themselves by pressing a lever which caused
delivery of a 0.5 sec. train of 60/sec. impulses
through the implanted electrode. Under these
conditions stimulation of the amygdala or septum
produced response rates of 200-1000/hr. Stimu-
lation of the hypothalamus produced rates up to
6000/hr. Without stimulation, rates of lever
pressing were below 25/hr. Reserpine and chlor-
promazine decreased rates of lever pressing for
stimulation in animals with electrodes in the
hypothalamus and amygdala by one-half or more;

sir

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SHER-
llinois

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ording
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March 1956

in some animals the response was blocked com-
pletely. In animals with a septal placement the
depression of response rate was markedly less;
and in most animals the response rate was little
altered at times when animals with amygdala or
hypothalamic placements had ceased to respond.

1455. Drug therapy of hypertension in the
elderly patient. Sam Kinarp, Epwarp Dern-
NIS AND RoBERT HERSCHBERGER (introduced by
RicuarD A. SErBERT). Baylor Univ. College of
Medicine, Houston, Texas.

This study is an evaluation of the response to
antihypertensive agents in elderly patients with
hypertension. The complications of hypertension
and response to drug therapy are compared to that
of the average hypertensive patient. Eighty-five
elderly patients were treated on an out-patient
basis using Rauwolfia, Rauwolfia plus hydralazine,
Rauwolfia plus pentolinium, or Rauwolfia plus
mecamylamine. The incidence of complications
was very high but comparison with a similar group
of average hypertensive patients revealed that
the only significant difference is the greater in-
cidence of electrocardiographic abnormalities in
the older patients. Thirty-four of the older pa-
tients were treated with Rauwolfia alone and the
response rate was less than in the average patient.
Side effects were similar in both groups. Fourteen
elderly patients were treated with Rauwolfia
combined with hydralazine. The response rate was
similar to the average hypertensive patient but
the incidence of patients becoming normotensive
was lower in the older group. Side effects were
similar except for headache and palpitations which
were more common in the older patients. Seven-
teen patients treated with Rauwolfia plus pen-
tolinium responded in about the same percentage
as the average patients. Of the 20 patients treated
with Rauwolfia plus mecamylamine, one-half
returned to normotensive levels and the over-all
response was the same as in the average patient.
It is concluded that the centrally acting anti-
hypertensive agents are less effective in the
elderly patient than in the average hypertensive
patient but when the more potent ganglionic
blocking agents are used, reduction in blood
pressure is equally as effective in both groups of
patients.

1456. Metabolism of 2,5,8-trimethylchromone.
J. Stanton Kine, Jr., Norman H. LEAKE
AND Ne.tta H. Warnock (introduced by Liuoyp
W. Hazueton). Research Labs., S. E. Messengill
Co., Bristol, Tenn.

The metabolism of the synthetic coronary
vasodilator 2,5,8-trimethylchromone was studied.
In humans, absorption of a 250-500 mg oral dose
began almost immediately, since chromone-
positive material appeared in the urine after only

PXUM

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

447

25 min. In 3 experiments excretion of such material
continued from 15-38 hr. Free 2,5,8-trimethyl-
chromone comprised only a negligible fraction of
such material. Evidence was found for at least
3 metabolites, which, on the basis of a charac-
teristic color test, were 2-methylchromone de-
rivatives. Two of these were apparently excreted
as glucuronides. One metabolite was isolated and
partially identified. It accounted for about 50%
of the orally ingested 2,5,8-trimethylchromone.
In rats, absorption of an oral dose of the drug
occurred from the stomach. The drug, when given
intravenously, was rapidly removed from the
blood of intact animals. No preferential site of
absorption or storage was found in animal tissues
and fluids such as the liver, brain, pancreas, fat
and bile.

1457. Reaction of lymphocytic system to
lethal an sublethal doses of alcohol.
Gerba I. KLINGMAN AND Gorpon R. HENNIGAR
(introduced by Harvey B. Haaa). Depts. of
Pharmacology and Pathology, Med. College of
Virginia, Richmond.

The oral (25%) and intravenous (20%) admin-
istration of lethal and sublethal doses of alcohol
(12 and 8 ml of absolute alcohol/kg, respectively)
to unanesthetized dogs produced changes of the
lymphocytic tissues. These changes consisted of
necrosis of germinal follicles throughout the entire
lymphocytic system. Hemorrhagic gastritis,
particularly of the distal part of the stomach, was
noted after oral administration. Occasionally loss
of lipoid in the deeper layers of the adrenal zona
fasciculata was observed. Continuous intravenous
infusion of epinephrine or norepinephrine to
alcoholized dogs did not appear to intensify this
reaction. These changes did not occur in
adrenalectomized dogs. The intravenous ad-
ministration of multiple doses of hexamethonium
chloride (3 mg/kg) appeared to reduce them. The
reaction of the lymphocytic system to alcohol in a
number of other species has also been investigated
as well as the effect of ether and pentobarbital in
dogs. The possible mechanism is discussed by
which this reaction of the lymphocytic system to
alcohol is produced. (Supported by a grant from
the Division of Alcohol Studies and Rehabilita-
tion, Department of Health, Commonwealth of
Virginia.)

1458. Production of tolerance to anticon-
vulsant effect of acetazoleamide. ALAN
Kocu* anp Drxon M. Woopsury. Dept. of
Pharmacology, Univ. of Utah, College of Medicine,
Salt Lake City.

Both the inhalation of carbon dioxide and the
administration of acetazoleamide alter the
pattern of maximal electroshock seizures in rats.
During chronic treatment with acetazoleamide,

448

tolerance develops to the effects of both the
inhibition of carbonic anhydrase and the in-
halation of carbon dioxide. Thus, the initial
ED» of acetazoleamide is 2 mg/kg, while after only
2 administrations at 48-hr. intervals it is increased
to 10 mg/kg. Concurrently, the EDs of carbon
dioxide, as measured in a gas mixture containing
20% oxygen, rises from 2.2 to 5%. Continued
administration of acetazoleamide fails to elevate
further the anticonvulsant EDs of either agent.
It may be presumed that both carbon dioxide and
acetazoleamide mediate their anticonvulsant
action through a common mechanism involving
carbonic anhydrase. This mechanism is ap-
parently unrelated to the regulation of cellular
acid-base balance, for anticonvulsant doses of
acetazoleamide produce no detectable changes in
brain intracellular pu. The effects of the inhalation
of carbon dioxide and of the administration of
acetazoleamide on acid-base balance and elec-
trolyte composition in brain cells will be discussed.
(Supported in part by contract number 18(600)921,
USAF and Fellowship number HF-5630, Natl.
Insts. of Health, PIIS.)

1459. Toxicity of 1,2-dibromo-3-chloropro-
pane. Jrro K. Kopama anp Mary K. Dun tap
(introduced by Cuartes H. Hine). Dept. of
Pharmacology and Exptl. Therapeutics, Univ. of
California, San Francisco.

A series of toxicity tests were carried out on
albino rabbits and Long-Evans rats to determine
the relative toxicity of 1,2-dibromo-3-chloropro-
pane (Nemagon) as compared with other, stand-
ard fumigants. Primary irritation studies in-
dicated a minimal irritating effect on rabbit skin
and moderate irritation of the eye without corneal
scarring. Twenty cutaneous applications of 0.25
gm had no effect on the skin of rabbits other than
slight crusting after the fifteenth application. The
percutaneous LD» was 1.4 gm/kg. A 90-day feeding
experiment showed Nemagon to be much less toxic
than DDT or Chlordane, possibly because of
more rapid metabolism and minimal storage in the
body. When groups of 14 male and 14 female rats
were fed 5; 20, 150, 450, and 1350 ppm, the lowest
level causing gross effects (retarded growth) was
150 ppm for females and 450 ppm for males. Liver
and kidney weights were significantly increased in
females fed 450 ppm but males showed a difference
only at 1350 ppm. There were no deaths attribut-
able to the compound except at 1350 ppm, where
mortality exceeded 50%. Inhalation studies
showed favorable comparison with other common
fumigants. Fifty exposures of groups of 15 male
rats over 10 wk. to 5, 10, 20, and 50 ppm caused no
apparent visceral damage and no significant
differences in organ/body weight ratios; growth
was significantly retarded except at 10 ppm. Three
of 15 rats died in the 50-ppm group. LCs values

FEDERATION PROCEEDINGS

Volume 16

for 1, 2, 4 and 8 hours were 368, 232, 154, and 103
ppm, respectively. Acute exposure to 135-670
ppm caused pulmonary irritation and hepatic
damage with minimal hypnotic effect. (Supported,
in part, by a grant from the Shell Develop. Co.)

1460. Comparison of effects of chlorpromazine
and secobarbital sodium on certain psy-
chological tests. Conan KorRNETSKY (intro-
duced by J. Cocurn). Natl. Inst. of Mental
Health, Natl. Insts. of Health, Bethesda, Md,
The psychological effects of 200 mg of seco-

barbital sodium (seconal) and 200 mg of chlor-
promazine were compared in normal young adults.
Each subject received the 2 drugs as well as 2
placebos on separate days. The drugs and placebos
were taken orally in identical capsules. The
‘double-blind’ technique was employed through-
out. Seventy-five min. after the drugs were in-
gested, a battery of tests was administered. The
tests consisted of the following: speed of addition
(3 digits and 9 digits), speed of copying digits,
digit symbol test, pursuit rotor test, tachisto-
scopic size discrimination, and threshold for
touch. The total time to complete all tests was
approximately 90 min. By means of converting all
scores to a percentage of the mean of the 2 control
scores, an over-all performance score was ob-
tained. The total performance score of subjects
receiving chlorpromazine and seconal was sig-
nificantly different (P < .05 and .01 respectively)
from the total performance score of subjects re-
ceiving placebos. Despite this over-all signifi-
cance, chlorpromazine had significant effects
(P < .01) on only the pursuit rotor test, a test of
motor coordination. Individual test analyses re-
vealed statistically significant differences be-
tween the performance of the subjects on seconal
and placebos on the tests of addition, digit symbol
and pursuit rotor. Although seconal produced
greater impairment than did chlorpromazine, this
difference was not statistically significant. In no
individual test was the impairment of performance
caused by seconal significantly greater than that
caused by chlorpromazine.

1461. Effects of various cardiac stimulants
and cardiac depressants on potassium
exchange in isolated perfused rabbit heart.
B. Korou* ann K. I. MELviItie. Dept. of Phar-
macology, McGill Univ., Montreal, Canada.
Using radioactive K*? the exchange rates of po-

tassium in isolated rabbit hearts perfused with

normal Locke were calculated from the differences
between the amounts of K* in the perfusion
fluid and in the collected perfusates. The rates of
coronary inflow and heart contractions were also
recorded concomitantly; and, the net gain or loss
of potassium (K*) from the heart simultaneously
determined from analyses of the perfusion fluid

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March 1956

and collected perfusates. Following injections
(3 experiments with each agent) of epinephrine
(10 wg), norepinephrine (10 wg), isoprotenol (1
yg) and aminophylline (5 mg), there was a sig-
nificant net loss of potassium, and marked in-
creased turnover (20-30 fold) of K* associated
with the cardiac stimulation. Since there is an
overall loss of potassium from the heart in each
case, it is concluded that the predominant effect
of these agents is to increase the outward phase of
potassium exchange. Conversely, following in-
jections (3 experiments with each agent) of
acetylcholine (100 ug) and methoxamine (10 mg)
there was a significant net uptake of potassium,
but again a similar increased potassium turnover
associated with the cardiac depression, sug-
gesting that the predominant effect of these latter
agents is on the inward phase of potassium ex-
change. It has been previously shown that when
the potassium in the perfusing fluid is decreased
cardiac stimulation occurs, and conversely,
myocardial depression is associated with increased
potassium. It is therefore postulated that stimu-
lation of the heart contractions by the drugs
studied might be due to the observed increased
potassium turnover with net loss and conversely,
depression of the heart contractions by the agents
studied, might be due to the observed net gain of
potassium. (Supported by a grant from the Natl.
Research Council of Canada.)

1462. Chronotropic cardiac action of reser-
pine. Orto KRayeR AND JAIME FUENTES.*
Dept. of Pharmacology, Harvara Med. School,
Boston, Mass.

In the heart-lung preparation of the dog (HLP),
with a blood volume of about one liter, reserpine
in a dosage range of 0.01-5 mg causes a heart rate
increase. Within this range the positive chrono-
tropic action and the rate of development of
maximal effect are proportional to the dose.
Further increase of the dose decreases the heart
rate which, at dose levels of 20 mg or above, may
drop below the initial rate. Veratramine an-
tagonizes the heart rate increase caused by
reserpine. The positive chronotropic action of
epinephrine and ephedrine in the HLP is an-
tagonized by reserpine. The inhibition of cardiac
acceleration occurs in the presence as well as in
the absence of atropine. In this regard, the cardiac
action of reserpine resembles that of veratramine.
When maximal acceleration of heart rate was pro-
duced by 2-3 mg ephedrine (KRAYER AND OuRIS-
son, J. Pharmacol. & Exper. Therap. 112: 341,
1954), the dose of reserpine needed to cause 50%
inhibition of acceleration was 15 mg (range 8-20
mg in 5 experiments). On a molar basis, the
potency of reserpine is appruximately y's of that
of veratramine. As in the case of veratramine,
pretreatment of the HLP (with 25 mg reserpine or

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

449

more) does not prevent, but markedly reduces, the
positive chronotropic action of epinephrine or
ephedrine. These experiments bear out Salva’s
assumption, based on experiments in the spinal cat
under epinephrine or norepinephrine, that reser-
pine has antiaccelerator activity. (Actualités
Pharmacologiques ed. R. Hazarp, Paris, 1955, p.
153).

1463. Influence of tension and neuromuscular
blocking agents on tetanus. W. L. Kunn*
AND E. F. VAN MAANEN. Dept. of Pharmacology,
Univ. of Cincinnati College of Medicine, Cin-
cinnati, Ohio.

The intact rat sciatic-gastrocnemious prepa-
ration was stimulated 10 sec. every min. at a
frequency of 500/sec. Contractions were recorded
by means of a strain gauge and an ink-writing
oscillograph. Increments of 100 gm tension dis-
placed the cantilever 0.5 cm. The maximal short-
ening of the muscle during tetanus increased as
resting tension was increased up to 100 gm. Fur-
ther increase in resting tension had little influence
on the maximal tetanus height. The occurrence of
the first depression, i.e. stage 2 (RoSENBLUETH
AND Cannon, Am. J. Physiol. 130: 205, 1940)
depended on the resting tension. Below 40 gm
it was prominent; above 80 gm it was not percep-
tible. Intravenous tubocurarine, decamethonium
or neostigmine decreased the height of the second
peak (3a), the second depression (3b) and the
third peak (8c) at doses which did not affect the
first peak (stage 1). The ppDs0’s of decamethonium
and tubocurarine for stage 1 were 5-9 times the
PD50’s for stages 3a, 3b, and 3c. With neostigmine
the PDs» for stage 1 was 180-210 times those of the
latter stages. The extreme sensitivity of stages
3a, 3b, and 3c with respect to stage 1 after neo-
stigmine indicates the importance of the cholin-
esterase system in tetanus. Tubocurarine never
augmented the tension at stage 3b. Since stage 3b
is sensitive to both tubocurarine and deca-
methonium, excessive depolarization cannot be
its primary cause.

1464. Inhibition of influenza A virus in tissue
culture by canavanine flavianate. W. D.
Kunp1n,* M. L. Ropsins* anp Paut K. Smira.
Dept. of Pharmacology, George Washington Univ.
School of Medicine, Washington, D. C.
Canavanine flavianate was effective in inhibiting

the growth of influenza A virus (WS strain) in

tissue cultures of dispersed chick embryo lungs.

The tissues grew in concentrations of up to 0.5

mg of canavanine flavianate per ml. Virus growth

was inhibited at concentrations down to 0.03

mg/ml. Flavianic acid had no inhibitory effect.

Canavanine sulfate had an index of but two.

Inhibition of the virus and toxicity to the tissue

by canavanine sulfate or canavanine flavianate

450

were reversed competitively by l-arginine, but
not by d-arginine, ornithine citrulline, glutamic
acid, proline or hydroxyproline. Canavanine
flavianate and flavianic acid were found to be
equally virucidal. Canavanine sulfate showed no
virucidal effects. When put in the diet of mice
infected with influenza A, canavanine flavianate
at a concentration of 0.5% failed to alter the
course of infection. Other investigators in this
laboratory found canavanine sulfate slightly
effective and canavanine flavianate markedly
effective in inhibiting the poliomyelitis virus in
monkey kidney tissue culture and in HeLa cell
tissue culture.

1465. Biochemical effect of reserpine on sero-
tonin binding sites. R. KuntzMan,* SIDNEY
UpENFRIEND, E. G. Tomicu,* B. BropiE AND
P. A. SHore.* Lab. of Chemical Pharmacology,
Natl. Heart Inst., Bethesda, Md.

The concept has been presented that the ad-
ministration of reserpine causes a change in the
serotonin binding capacity of various tissues,
including brain, intestines and platelets. As a
result serotonin is released from tissues and
metabolized. Serotonin continues to be formed
but the serotonin binding sites are impaired. This
concept would have more validity if it could be
shown that reserpine impaired the ability of
animal tissues to take up serotonin available in
relatively high concentration. The present studies
show that blood platelets, isolated from guinea
pigs 16 hr. after the administration of 5 mg/kg
reserpine, show a marked deficiency in their
ability to take up serotonin, compared to platelets
from normal animals. Moreover, administration
of 5-hydroxytryptophan, the precursor of sero-
tonin which yields the amine by decarboxylation,
causes a marked increase in brain serotonin in
normal rabbits, but fails to do so in animals 16
hr. after they had been given 5 mg/kg reserpine.
5-Hydroxytryptophan decarboxylase, the enzyme
that catalyses the formation of serotonin is not
affected by reserpine. Since reserpine could no
longer be detected in brain, the drug appears to
induce an irreversible alteration in the serotonin
binding sites. As a consequence serotonin, though
still being formed, remains free and exerts its
pharmacologic effect until new binding sites are
formed.

1466. Continuous control of alveolar pCO, in
man in studies of meperidine-induced
respiratory depression. C. J. LAMBERTSEN
AND H. WENDEL. Dept. of Pharmacology, Univ.
of Pennsylvania School of Medicine, Philadelphia.
A method for automatically or manually im-

posing desired elevated levels of alveolar pCO,

upon human subjects and maintaining these levels

FEDERATION PROCEEDINGS

Volume 15

in spite of respiratory alterations was devised.
This method, studied up to 46 mm Hg, permits
control of end-tidal pCO: within +1.0 mm Hg in
most subjects, within +0.5 mm Hg in some.
Without such control, changes in pCO: accom-
panying ventilatory responses to drugs or altered
physiological states introduce new variables and in
themselves limit the magnitude of primary effects
upon respiration and circulation. Thus, in subjects
breathing air without controlled alveolar pCO,
150 mg meperidine i.m. produced an average,
nonsignificant decrease in ‘steady state’ respir-
atory minute volume (RMV) of 6.4%. When
respiration was driven by CO2 administration
without pCO, regulation, this meperidine dose
significantly decreased ‘steady state’ RMV 61.4%
at an interpolated 46 mm Hg alveolar pCO,
(J. Pharmacol. & Exper. Therap. 108: 376, 1953).
In the present investigation, continuous control
of alveolar pCO, at 46 mm Hg allowed direct,
continuous measurement of undamped respir-
atory response to meperidine, revealing rate of
depression and its degree at intervals after drug
injection. Using this method, it was found from
average results in 6 subjects that 100 mg meperi-
dine/70 kg i.m. began to reduce RMV within 15
min., produced maximal depression of 61% within
107 min., exerted a mean depression over 4 hr.
of 55% and, at 4 hr., still depressed RMV 37%.

1467. Relation between cerebral synaptic
actions of mescaline, indoles and tran-
quilizers. THomas W. Lanaritt,* E. Ross
Hart AND AmMEDEO 8S. Marrazzi. Neurology
Branch and Clin. Research Div., Chemical Corps
Med. Labs., Army Chemical Ctr., Md.

The authors have described a cerebral cortical
synaptic inhibitory action of mescaline as de-
termined by the reduction in transmission of the
impulse initiated by a test stimulus delivered
through the transcallosal association pathway of
the nembutalized cat and recorded electrically at
the surface of the contralateral optic cortex. This
experiment was suggested by the structural
similarity of mescaline to adrenaline, whose
cerebral synaptic inhibitory action had been pre-
viously described by this group. Since adreno-
chrome is an indole related to and derived from
adrenaline, it was also studied along with seroto-
nin, an indole natural to the brain and already
found by us to be a more potent inhibitor than
either mescaline or adrenaline, and bufotenine, a
dimethyl serotonin. The relation of these to tran-
quilizers and particularly to reserpine, because of
its relation to both the adrenolytic yohimbine and
to mescaline, was examined. Predictions on the
basis of structure are partly borne out in that the
chemically similar compounds all have an effect
on cerebral synaptic transmission. They either

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March 1956

inhibit, as in the case of adrenaline-like structures
and somé indoles, or block the mescaline in-
hibition, presumably on a competitive basis, as
is true for other indoles and the tranquilizers
(reserpine, chlorpromazine and _ azacyclonol).

1468. Can psychiatric patients be used for
evaluating hypnotic drugs? Louris LAsaAGNa
AND JOHN ImBopEN.* Medicine, Pharmacology
and Psychiatry Depts., Johns Hopkins Med.
School, Baltimore, Md.

Ward nurses at the Phipps Clinic routinely
make rounds on their patients every 30 min.
throughout the night, recording whether each
patient is asleep or awake. This seemed to pro-
vide a superb opportunity to evaluate the sleep-
producing effects of drugs. Accordingly, 27
severely neurotic or psychotic patients, who were
considered to have sleep problems by the medical
staff and who were not receiving major specific
therapy during the week’s period required for the
experiment, were chosen for study. Each patient
was given, on consecutive nights, in random
order, the following medicaments, all made up in
elixir form so as to be essentially indistinguish-
able one from the other: placebo; chloral hydrate
(CH) 1 and 2 gm; trichloroethanol (TCE) 1
and 2 gm; and pentobarbital sodium (PE) 0.15
and 0.3 gm. All medications were identified by
code letters, and the code frequently changed.
Neither patients, nurses nor technicians were
aware of the-nature of the various solutions. In
the mornings, the patients were asked a series of
questions by either a technician or nurse on their
subjective evaluation of hypnotic and side effects.
Results: All medications were significantly better
than placebo in inducing sleep. The higher doses of
CH and TCE and both doses of PE were sig-
nificantly better than placebo in maintaining
sleep. By most criteria, the higher doses of PE
and TCE (in that order) yielded best results. An
interesting finding was the tendency for subjec-
tive reports by the patients to underestimate
hypnotic effects (when compared with the nurses’
descriptions).

1469. Hormonal influences upon arterial
medionecrosis. Davip LEHR, CONSTANCE
Martin* AND Rospert Mitora.* Dept. of

Pharmacology, New York Med. College, Flower

and Fifth Ave. Hosps., New York City.

It was shown (Lehr, et al. Federation Proc.
13: 1954) that adrenalectomized rats maintained
on saline were unable to survive for more than a
few hours the stress of the procedure required for
the production of renogenic arterial medione-
crosis. Emergence of the arterial injury was made
possible by pretreatment with a combination of
cortisone and DCA. Administration of DCA alone
did not effect substantial extension of the lifespan

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

451

following the standard renal injury, whereas
cortisone alone resulted in adequate survival
without the appearance of arterial lesions. Using
a subcutaneous modification (LEHR, et al. this
issue), which proved less stressing than the
intraperitoneal introduction of the nephrotoxic
agent, the results, except for better overall
survival, compared closely with those reported
above. Similar observations were made in hy-
pophysectomized rats. Unprotected, the majority
succumbed in shock within 24 hr. following the
intraperitoneal injection of the nephrotoxic agent.
With the cortisone-DCA combination good
survival was achieved and typical arterial lesions
were induced. As in adrenalectomized rats, DCA
alone failed to extend survival, while cortisone
alone, in a preliminary study, permitted adequate
prolongation of life without the appearance of
vascular lesions. Since parathyroid deficiency was
shown to prevent the development of experi-
mental muscular necrosis in arteries and else-
where (LEHR AND Martin. J. Pharmacol. In
press), adrenalectomized cortisone treated rats
were given parathyroid extract in excess, Pre-
liminary results indicate that arterial medio-
necrosis can be produced under these conditions.
(Supported by Research Grant No. H-890 (C4)
from the Natl. Heart Inst., Natl. Insts. of Health,
PHS.)

1470. Modification of method for production
of cardiovascular and smooth muscle
necrosis in the albino rat. D. Lenr, R.
Mitora* anp C. Martin.* Dept. of Pharma-
cology, New York Med. College, Flower and
Fifth Ave. Hosps., New York City.

Our standard procedure for the production of
cardiovascular and smooth muscle necrosis in the
albino rat calls for the intraperitoneal injection
of sodium acetyl sulfathiazole (NaAST) for the
purpose of inducing obstructive nephropathy
(LEHR AND Cuure. J. Mt. Sinai Hosp. 19: 106,
1952). While the stress of peritoneal irritation was
well tolerated by the intact rat, it appeared to be
an important contributory cause to the early
death of adrenalectomized and hypophysecto-
mized animals. To overcome this difficulty, a
subcutaneous modification of the procedure was
developed which proved far less stressing, as was
apparent from the behavior of the intact rat and
especially from the extended survival of the
adrenalectomized rat. In animals weighing about
400 gm, a dose of 1.0 gm NaAST/kg body weight
proved highly satisfactory, both with regard to
survival and to incidence of cardiovascular lesions.
Upon completion of the subcutaneous administra-
tion, the animals appeared to suffer no discomfort
and did not show the severe depression and
peculiar ‘stretching’ gait seen regularly after
intraperitoneal injection. They also lacked the

452

initial hypotension ascribed to peritoneal irrita-
tion and fluid extravasation. Hence, there was an
earlier onset of hypertension, occurring often
within 48 hr., and in conjunction with it a speed-
up in the emergence of severe vascular damage.
Thus, for animals intolerant of severe stress, the
subcutaneous administration has distinct ad-
vantages over the intraperitoneal introduction of
the noxious agent. In addition, injection under
constant visual control is foolproof, eliminating
inadvertent introduction into the gut or injury
to abdominal organs. (Supported by research
grant No. H-890 (C4) from the Natl. Heart Inst.,
Natl. Insts. of Health, PHS.)

1471. Experimental auricular fibrillation in
the thyrotoxic dog. Pxaruuie E. LEvEQUE
(introduced by R. P. Auntautst). Dept. of
Pharmacology, Med. College of Georgia, Augusta.
Although hyperthyroidism has long been known

to produce cardiac hyperirritability, until now

no one has been able to demonstrate successful
induction of the ‘Thyrocardiac’ state in laboratory

animals. Surtshin and Rucknagel (Am. Heart J.

45: 781, 1953) report that it cannot be ac-

complished. Long term thyroid extract feeding

and adequate acetylcholine administration by
intravenous injection have been found to provoke

auricular flutter and/or fibrillation in 13 of 16

dogs. Many of the dogs showed this sensitivity to

acetylcholine for long periods of time, but the
usual reaction was an acute hypersensitivity
around the 7th to 10th day with less or no sen-
sitivity to acetylcholine before this time and in-
creasing refractoriness after this sensitive period.

Experiments with purified thyroid hormone are

continuing for the purpose of investigating the

possibility of acute production of this sensitivity
and to elaborate the usefulness of this technique.

(Supported in part by a grant from Parke, Davis

and Co. to Dept. of Pharmacology, Univ. of

Oregon Med. School.)

1472. Cerebral circulation. Haroutp D. GREEN,
L. Earu Watts* anp N. Max Lewis.* Dept. of
Physiology and Pharmacology, Bowman Gray
School df Medicine, Wake Forest College, Winston-
Salem, N.C.

Communications between intracranial and
extracranial arteries in the dog were identified
and evaluated by means of dissection, perfusion
studies and post-mortem angiograms (barium-
gelatin). The right common carotid artery was
exposed and all branches in the cervical region
proximal to the bifurcation ligated. By resection
of the right mandible and orbital structures, the
external carotid and internal maxillary arteries
were exposed. Ligation of the latter and its
branches in the orbit interrupted the anastomotic
channels between this artery and the intra-

FEDERATION PROCEEDINGS

Volume 1§

cranial circuit. Since interruption of the com-
munication between the occipital artery and the
intra-cranial circuit was not feasible, this artery
was cannulated and perfused at aortic pressure
as was the segment of external carotid between
the bulb and the middle meningeal. By means of
these procedures it is felt that changes in intra-
cranial vasomotor tone can be measured unin-
fluenced by extra-cranial vasoactivity. Blood
flows at aortic pressure in the right internal
carotid and middle meningeal arteries were regis-
tered simultaneously by means of separate elec-
tromagnetic flowmeters. In 5 satisfactory ex-
periments mean control flow was 2.3 ml/min. in the
former and 8.5 ml/min. in the latter. Intra-
cranial epinephrine caused constriction which was
greater in the middle meningeal than in the
internal carotid bed; but less than that in most
systemic beds. Isopropyl levarterenol caused a
moderate dilatation which was approximately
equal in both vascular beds. (Supported by
L.I.M.R.F.)

1473. Comparison of the diuretic activity of
pyrogen with that of the urinary diuretic
factor. J. MAXwELu LittLe. Dept. of Physi-
ology and Pharmacology, Bowman Gray School of
Medicine, Wake Forest College, Winston-Salem,
N.C,

Previous studies have demonstrated the pres-
ence of a substance in dog, human and pregnant
mare urine which is diuretic when tested in the
dog (Am. J. Physiol. 180: 173, 1955). The pos-
sibility that pyrogen might be diuretic and that
the diuresis of the urinary factor might be due to
pyrogen has been considered. Pyrogen (Piromen)
was found to be diuretic in a dose as low as 0.4
ug/kg. Attempts have been made to destroy the
diuretic activity of either the urinary factor or
pyrogen without affecting the activity of the other
preparation. Treatment with hydrogen peroxide
and heat, and heating in a buffer at px 3.0 for
varying times were unsuccessful, since the diuretic
activity of both the urinary factor and pyrogen
was destroyed by these treatments. Heating the
urinary factor in a buffer at pa 10.0 for 1 hr. ina
boiling water bath destroyed its diuretic activity,
whereas the same treatment of pyrogen in com-
parable doses had little effect on its diuretic
activity. It is concluded that the diuretic activity
of the urine preparation cannot be explained on
the basis of the presence of pyrogen. (Supported
in part by Grant No. H-1998, Natl. Insts. of
Health, PHS.)

1474. Potentation of heparin anticoagulant
action with sulfonated lignin. Trp A.
Loomis. Dept. of Pharmacology, School of Med-
icine, Univ. of Washington, Seattle.

Sulfonated lignins were previously shown to

E XUM

lume 16

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March 1956

have a heparin-like anticoagulant action. One of
the fractions (lignin-3) of the total sulfonated
lignins prepared from commercial waste sulfite
liquor was further investigated for its anticoagu-
lant action when used in combination with hep-
arin. Heparin (0.5 mg/kg) plus lignin-3 (5 mg/kg)
resulted in a 2-fold more intense anticoagulant
action than that which could be produced by the
same dose of either preparation alone. The du-
ration of anticoagulant action following a single
intravenous injection was 4-5 hr. Jn vitro experi-
ments demonstrated that the ratio of the con-
centration of lignin to that of heparin which re-
sulted in maximum potentiating anticoagulant
effect is approximately ten. The potentiating
effect of lignin is evident only when the heparin
concentration is adequate to produce a 1} to 2-
fold increase in clotting time. Whole fresh blood,
in which the clotting time was prolonged to
greater than 20 times normal by the addition of
heparin plus lignin, produced a firm clot im-
mediately following the addition of purified
thrombin.

1475. Drugs inhibiting influenza virus, strain
A, PR8, in tissue culture. GERTRUDE S. Lum*
AND Pau K. Situ. Dept. of Pharmacology,
The George Washington Univ. School of Med-
icine, Washington, D. C.

A tissue culture system with chick chorioal-
lantoic membrane grown directly on glass tubes
and the tubes observed for somatic cell outgrowth,
was used to test several hundred drugs for their
ability to inhibit growth of influenza virus, strain
A, PRS8, as established by lack of hemagglutina-
tion after 44- to 48-hr. growth. Four thiosemicarba-
zones and 6 dipheny] and related compounds were
found to have a therapeutic index of over 8. The
mode of drug action was studied further by testing
the in vitro effect of the drugs on the virus, pre-
vention of adsorption of virus to cell, effect on
hemagglutination and effect on cell respiration.
The action of no drug was so explained. In all cases
the number of virus particles was considerably
less in the tissues in treated tubes than in controls
as evidenced in experiments designed to show
whether action of the drug prevented release of
virus particles from the cells and by studies of
growth curves. Preliminary studies show reversal
of bis-(4-guanidinophenyl) methane sulfate and
bis-(4-guanidinophenyl) sulfide by arginine and
4-nitrodiphenylamine by niacin. Further work is
being done on this phase to determine whether
these reversals of inhibitors are competitive.
Of 7 drugs tested by oral feeding, beginning 1 day
prior to infection, bis-(4-guanidinophenyl) meth-
ane sulfate provided appreciable protection, and
a,a’ dipyridyl, p-hydroxy-benzaldehyde, 3-thio-
semicarbazone, and a-amylcinnamaldehyde, 3-

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

453

thiosemicarbazone provided moderate protection.
All controls in this experiment died within 8 days.
Further studies of the therapeutic effect of these
drugs are in process.

1476. Influence of newer oral cardiac
diuretics. Davin I. Macurt. Div. of Pharma-
cology, Labs. Sinai Hosp., Baltimore, Md.
The author reported previously (Am. Heart J.

31: 460, 1946) that several mercurial diuretics em-
ployed in cardiology exerted marked thrombo-
plastic action on the blood of animals, and this
finding may explain occasional serious accidents
after their use. That study stimulated the present
investigation of some newer oral nephrotropic
agents now in use. The compounds examined were
acetazoleamide, aminometramide and _ chlor-
merodin. The 3rd is an organic mercury com-
pound. All experiments were performed on rabbits
administering the drug through stomach tube.
Extensive previous experiments established rab-
bits as a reliable test animal for study of blood
coagulation (Antibiotics and Chemotherapy, 3:
990, 1953). Clotting was measured by Macht and
Hoffmaster’s method, (Science 115: 91, 1952),
coagulation time being determined before ad-
ministration and 1, 2 and 3 hr. after the drug;
also on the next day. Large doses of the com-
pounds, 10-20 times that for humans were em-
ployed with no toxic effects. Acetazoleamide
produced no change in coagulation time; neither
did aminometramide. Chlormerodin occasionally
showed decrease in coagulation time an hour later,
but always with recovery to normal at the end of
the day or by next morning. This transient
shortening in clotting time was probably due to
the very large doses employed, indicating that
the mercury is in firm organic combination and
practically undissociated.

1477. Arrhythmias induced by epinephrine
and norepinephrine in dogs during the
first fifteen days following coronary liga-
tion. Harriet M. Maine anp Neri C.
Moran.* Lab. of Chemical Pharmacology, Natl.
Heart Inst., Bethesda, Md.

The anterior descending coronary artery of dogs
was ligated following the procedure of Harris
(Circulation, 1: 1318, 1950), and after the spon-
taneous delayed ectopic activity had subsided
the electrocardiographic responses to graded
equimolar doses of epinephrine and norepineph-
rine were determined. Both epinephrine and
norepinephrine induced an exaggerated ectopic
activity in the postoperative period. This sen-
sitization to epinephrine and norepinephrine was
also conspicuous in 2 dogs in which a descending
branch of the circumflex artery was ligated. The
ectopic activity took the form of AV nodal
rhythms, runs of ventricular rhythms with alter-

454

nating periods of complete AV dissociation and in-
terference dissociation and periods of normal sinus
rhythms with scattered ventricular extrasystoles.
The total number of ectopic beats in the 2-min.
period following the beginning of each injection
was proportional to the logarithm of the doses of
epinephrine or norepinephrine both before and
after ligation. Following the operation there was
a 2- to 4-fold increase in sensitivity to each drug.
Norepinephrine produced longer lasting arrhyth-
mias and usually a higher maximum rate of
ectopic rhythms than the corresponding equi-
molar dose of epinephrine. These sensitized
ectopic responses to epinephrine and norepineph-
rine were almost completely abolished by atro-
pine, suggesting either exaggerated reflex vagal
activity following coronary ligation, or masking of
ectopic activity by the high sinus rate resulting
from the atropine.

1478. Effect of 6-mercaptopurine on nucleic
acid synthesis. H. GrorGE MANDEL, JOSEPH
K. Inscoz,* Harriet M. Maina aNnp PAvL
K. Smitu. Dept. of Pharmacology, The George
Washington Univ. School of Medicine, Washing-
ton, D. C.

The growth of Escherichia coli was partially in-
hibited by the carcinostatic purine analog, 6-
mercaptopurine (6-MP), at a concentration of 3
mg/l. in glucose-salts media. The drug produced
no change in gross morphology of the organisms or
viability as determined by plate count. Simul-
taneous addition of 5 mg of guanine per liter of
medium partially reversed this inhibition. The
concentration of guanine was insufficient to supply
all the purines of the cell. Nucleic acids were
isolated by salt extraction from bacteria grown
with 5 mg/l. of guanine-4-C" in the presence and
absence of 3 mg/1. of 6-MP and were separated into
the purine fractions of RNA and DNA. The
relative specific activities of the adenine fractions
obtained from cultures grown in the presence of
6-MP were approximately 60% higher than those
of control cultures, whereas the corresponding
guanine fractions showed only a slight increase.
The total uptake of guanine-C" by the bacteria
in the presence and absence of 6-MP were identical
for the same extent of growth. Cells grown in the
presence of 6-MP had a smaller nucleic acid
content than did normal cells, as measured spec-
trophotometrically. The increase in relative
specific activities of the purines after 6-MP in-
hibition apparently is due to a decrease of de
novo purine synthesis without affecting direct
incorporation of preformed purines.

1479. Effects of prednisone, prednisolone and
fludrocortisone acetate on _ electroshock
seizure threshold. Leo F. Mansor,* Dorsry

FEDERATION PROCEEDINGS

Volume 16

EK. HotrKamp, ArTHUR EF, Heminc AND HowaRp

H. Curistran.* Research and Develop. Div.,

Smith, Kline and French Labs., Philadelphia, Pa.

Prednisone (0.2 and 0.6 mg/rat/day.), pred-
nisolone (0.3 and 0.8 mg/rat/day), and fludro-
cortisone acetate (0.3 and 3.0 mg/rat/day) were
compared with cortisone (1.0 and 3.0 mg/rat/day),
hydrocortisone (1.5 and 4.0 mg/rat/day) and
hydrocortisone acetate (0.3 and 3.0 mg/rat/day),
respectively, in the electroshock seizure threshold
test. Our procedure (Federation Proc. 11: 358,
1952) was an adaptation of the method described
by Woodbury (Rec. Prog. in Hormone Research
X: 65, 1954). Groups of 5 or 6 adrenalectomized,
adult male rats weighing 200-250 gm maintained
on 1% NaCl as the drinking fluid, were utilized for
each dosage level of treatment and for the con-
trol. The drugs were administered by single daily
subcutaneous injection using 10% ethanol-0.9%
saline as the vehicle. The change in threshold of
each animal was determined with reference to its
own pretreatment control. All agents lowered the
threshold, i.e. brain excitability was increased.
On a weight basis, prednisone and prednisolone
were each 2-4 times as potent as cortisone or
hydrocortisone; fludrocortisone acetate was
about 3 times as active as hydrocortisone acetate.

1480. Effect of lysergic acid diethylamide,
serotonin and related compounds on a
parasitic trematode, Fasciola hepatica.
Taa E. Mansour (introduced by E. Burp1n@).
Dept. of Pharmacology, Louisiana State Univ.
School of Medicine, New Orleans, La.

Lysergic acid diethylamide (LSD), in con-
centrations of 1 X 10-7 m or higher, increased the
amplitude, tone and rate of contraction of the
liver fluke, Fasciola hepatica, when motility was
recorded kymographically by the method of
Chance and Mansour (Brit. J. Pharmacol. 4: 7,
1949). Lower concentrations of LSD (5 xX 10°
to 5 X 10-8 m) temporarily reduced rhythmical
activity of the worm. Serctonin (4 X 10-* m) had
a stimulatory effect on the rate and amplitude of
contractions and on the tone. Following removal
of the central ganglia, isolated strips of the
parasite exhibited a similar response to the stimu-
latory actions of LSD and of serotonin. Epi-
nephrine, norepinephrine and histamine did not
affect the preparations even in high concentra-
tions. It was found that the flukes contain an
enzyme which catalyzes the oxidation of epineph-
rine and of tyramine, but not that of serotonin.
The latter inhibited the activity of this enzyme.
Fasciola hepatica metabolized carbohydrate at a
high rate. Production of propionic and acetic acids
(in an approximate ratio of 3:1) accounted for
almost all of the carbohydrate utilized anaero-
bically. Only 4-8% of the metabolized carbohy-

5. tab. OR... ae aes: - aie) Aen in | es iis (ee ck Se es

—_

ume 16

OWARD

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ia, Pa,

pred-
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March 1956

drate was converted to lactic acid. In the presence
of LSD (2 X 10-7 M) a 6-fold increase in the an-
aerobic production of lactic acid was observed
while formation of volatile acids was not changed
significantly. The effect of other sympathomi-
metic amines and of compounds related to lysergic
acid on the metabolism of the parasite will be
discussed. (Supported by Public Health Service
Grant * E-668 and by a grant from Swift and Co.)

1481. Diisonicotinoylhydrazine (DiINAH) in
the urine of mice and guinea pigs following
the administration of isoniazid (INAH).
R. W. MantTuHEr (introduced by J. M. Coon).
Dept. of Pharmacology, Jefferson Med. College,
Philadelphia, Pa.

Albert (Biochem J. 61: 125, 1955) has shown that
DiINAH is formed from INAH in the presence of
haemin in vitro. DiINAH was prepared by the
method of Albert: MP 259°; Rf of 0.70 on de-
scending chromatogram in butanol/water system.
Twenty-four hour urine samples were collected
under toluene from guinea pigs and mice that had
received 10 mg/kg of C-labeled INAH intra-
peritioneally. Isotope dilution studies with
DiINAH on this urine show that 1-4% of the total
INAH-like material present was DiINAH. Urine
from guinea pigs generally contained more
DiINAH than that of mice. The DiINAH isolated
from the urine was recrystallized to constant
MP 260°; prior to the determination of its radio-
activity, purity was confirmed by paper chroma-
tography. Since even in the absence of haemin,
isoniazid can slowly be converted to DiINAH
in vitro, the DiINAH found may not have been
formed in the tissues but directly in the urine
from excreted isoniazid.

1482. Interrelationship between fat solu-
bility, duration of action and passage into
brain of various intravenous anesthetics.
Lester C. Mark, J. J. Burns, NATALIE TRov-
sor,* LEONARD Branp,* E. M. Papper AND
BERNARD B. Bropie. Dept. of Anesthesiology,
Columbia Univ. and New York Univ. Research
Service, Goldwater Memorial Hosp., New York
City, and the Lab. of Chemical Pharmacology,
Natl. Heart Inst., Natl. Insts. of Health, Bethesda,
Md.

High localization of a number of intravenous
anesthetics in body fat is associated, not only
with the transient action of small single doses, but
also with rapid passage across the blood-brain
barrier. In general, the fat/plasma ratio of bar-
biturates is enhanced by substitution of sulphur
for oxygen in the 2-position, by attaching an alkyl
group to nitrogen and by increasing the length
of 1 of the side chains. The fat/plasma partition
ratios, rates of biotransformation and rates of

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

455

passage into brain are compared for a number of
ultrashort-acting anesthetics including Surital,
Kemithal, Pentothal, Squibb 2365 (5-allyl-5(2,3-
bromocyclohexeny])-2-thiobarbituric acid), Evi-
pal and the non-barbiturate drug, Dolitrone
(5-ethyl-6-phenyl-m-thiazane-2-4-dione).

1483. Comparative pharmacology of various
carbamidines substituted with one to five
ethyl groups. Davip Fietpine Marsu. Div.
of Med. Sciences, McNeil Labs., Philadelphia,
Pa.

Seven of the 8 possible carbamidine derivatives
having from 1 to all 5 of the available hydrogen
atoms replaced by an ethyl group have been pre-
pared and tested for pharmacological activity.
They have been compared with the chemically
related hexaethylguanidinium iodide, guanidine,
various alkylureas and xanthines. Although these
7 compounds have in common a low molecular
weight, similar chemical behavior and physical
characteristics, as well as identical components for
molecular architecture; they possess a wide
diversity of qualitative pharmacological activity
which has been confirmed in several animal
species. Many of the typical effects are not ob-
viously related to any of those produced by any of
the corresponding methyl analogs nor to the
parent substance. By far the most interesting
compound in the series is the only asymmetrical
tetraethyl carbamidine (McN-460) which is a low
toxicity, high potency, orally and parenterally
active diuretic. For example, 0.25-0.5 mg/kg i.v.
will double the urinary output of dogs. No tol-
erance, change in blood glucose level, cardiovas-
cular function, nor other action has been found
following oral administration to dogs of as much
as 200 mg/kg nor intravenous administration of
10 mg/kg. Similarly, as little as 0.1 mg/kg ad-
ministered to albino rats orally as a single dose or
given daily as repeated doses for as long as 6 wk.,
is effective, although doses as high as 900 mg/kg
are not lethal and produce slight drowsiness as the
only observable side effect. The exact biochemical
mechanism of this prolonged, relatively slow
onset, diuresis is not yet known. None of the other
carbamidine derivatives tested possess this un-
complicated diuretic activity.

1484. Differentiation of amino acid esterases
of rat skin extracts. CHARLES J. MARTIN
(introduced by T. K. T. Kruse). Biochemistry
Dept., Univ. of Pittsburgh School of Medicine,
Pittsburgh, Penna.

Rat skin extracts have been reported to contain

a complex spectrum of proteinase activities

(MartTIN, Federation Proc., 13: 260, 1954). Recent

work has shown that proteinase A can hydrolyze

N-acetyl-L-tyrosine ethyl ester (ATEE) and L-

456

tyrosine ethyl ester (TEE) and its activity against
both substrates can be stoichiometrically in-
hibited by an inhibitor (AIn) prepared from
blood. Proteinase A exists in both an active and
inactive state with maximal extraction of the
active enzyme from skin acetone powder occurring
at 1°C with a 1.6 m KCl solution. Another enzyme,
A,, is fully extracted with a 0.6 m KCl solution and
can hydrolyze ATEE but not TEE and can not be
inhibited by AIn. A water extract of skin powder
contains the maximal amount of As, an enzyme
which can hydrolyze TEE but not ATEE. The
AlIn preparation stimulates the TEE activity of
Ae and thus contains both an inhibitor for A and
an activator for A2. The differential extraction of
these enzymes, their characteristics, and relation-
ship to the previously reported proteinases B and
C will be discussed.

1485. Interrelationship of thymus and steroid
hormones in production of experimental
cardiovascular necrosis. CONSTANCE MARTIN*
AND Davin Lear. Pharmacology Dept., New
York Med. College, Flower and Fifth Ave. Hosps.,
New York City.

It was previously reported (MARTIN AND LEHR,
J. Pharmacol. & Exper. Therap. In press, 1955)
that thymectomy greatly increased the suscepti-
bility of 2-month old male albino rats to the
development of grossly visible necrosis of the
aortic media and the myocardium induced by ob-
structive nephropathy. In female rats, under the
same ccnditions, thymectomy was only moder-
ately effective in enhancing the extent of cardio-
vascular injury. The hypothesis was presented
that the thymus does not exert a directly protec-
tive action, but rather seems to suppress forma-
tion or release of an excess of certain steroid
hormones. The deleterious effect of thymectomy
is not obtained in the orchidectomized adult rat.
In line with this finding, androgen administration
in male weanling rats increases the incidence and
severity of cardiovascular lesions, while ad-
ministration of estrogens has the opposite effect
in female weanlings. These observations, pre-
viously presented in preliminary form, have been
confirmed in a large series of rats. More recently
it was found that the enhancing effects of thymec-
tomy and of ovariectomy upon the production of
eardiovascular necrosis cannot be elicited in
adrenalectomized animals maintained on cortisone
and saline. These and other data which will be
presented suggest that the thymus gland con-
tributes to the regulation of steroid hormone
activity. (Supported by research grant No. H-890
(C4) from the Natl. Heart Inst., Natl. Insts. of
Health, PHS.)

1486. Effect of chlorpromazine on cardio-

vascular responses to epinephrine and

FEDERATION PROCEEDINGS

Volume 15

norepinephrine in the cat. W. R. Martin

(introduced by K. R. Unna). Dept. of Phar-

macology, Univ. of Illinois College of Medicine,

Chicago, Ill.

It was previously reported that chlorpromazine
potentiated the inotropic, chronotropic and pressor
effects of norepinephrine and could either depress
or potentiate the effect of /-epinephrine. In order
to further study the difference between the effects
that chlorpromazine exerts on the vascular re-
sponses to epinephrine and norepinephrine a fixed
dose of norepinephrine (.5-1.0 yug/kg) or epi-
nephrine (1.0-2.0 ug/kg) was administered at 10-
min. intervals before and after the administra-
tion of chlorpromazine to spinal vagotomized
cats. Chlorpromazine enhanced and prolonged
the pressor response evoked by norepinephrine.
As a measure of the prolongation of the pressor
response to norepinephrine, the Decay Time 50
(the time necessary for the pressor response to
return to a value midway between the control
systolic pressure and the peak systolic pressure)
of all pressor responses was determined. A pro-
longation of the effects of norepinephrine by 60-
500% was observed within 5 min. after the ad-
ministration of chlorpromazine (5.0-10.0 mg/kg).
Two hr. after the administration of chlorpro-
mazine the effect of norepinephrine was still
prolonged by at least 30%. In contrast to its effect
on norepinephrine, chlorpromazine (1, 5 and 10
mg/kg) decreased or reversed the pressor response
to epinephrine in 11 of 12 cats. The action of
chlorpromazine on the vascular response to
epinephrine resembles that of an adrenergic
blocking agent such as Dibenzyline. However the
action of chlorpromazine on the vasomotor reac-
tion of norepinephrine cannot be explained by
adrenergic blockade. The possibility that chlor-
promazine interferes with the metabolism of
sympathomimetic amines will be discussed. (This
study was supported in part by Research Grant
983 of the Public Health Service.)

1487. Comparative effects of corticosterone
and cortisone on experimental coryne-
bacterium infection of mice. RicHarp C.
Mason,* Davin H. JoHNson* AND Harry J.
Rosinson. Merck Inst. for Therapeutic Research,
Rahway, N. J.

Several recent studies have suggested that
corticosterone (Compound B) does not possess
the same detrimental effect as cortisone (Com-
pound E) on host resistance to infection. It is the
purpose of this communication to present the
results of a comparison of the influence of corticos-
terone free alcohol and cortisone acetate on the
survival of mice experimentally infected by in-
halation with Corynebacterium pseudotuberculosis
murium (CPTM). Forty animals per group were
treated with a single subcutaneous injection

a gp a ee

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—

ume 15

[ARTIN
Phar-
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mazine
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March 1956

of steroid. The percentage of survival 10 days
after infection were: infected controls, 75%;
cortisone—1 mg, 58% and 10 mg, 3%; corticos-
terone—l1 mg, 73% and 10 mg, 33%. The gross
pathological lung lesions were also scored on a
scale of 1-5 at the time the animals were killed or
died. The relative mean values for each group
agreed closely with the respective percentage of
survival. From our results it appears that corti-
costerone was approximately } as potent as
cortisone acetate in suppressing the resistance to
CPTM infection in mice. The data demonstrate
no striking difference in effect of corticosterone
and cortisone acetate on infection when con-
sidered in terms of their relative glucocorticoid
activity.

1488. Response of normotensive and neuro-
genic hypertensive dogs to ganglionic
blocking agents and reserpine. R. A. Max-
WELL,* A. J. PLUMMER AND 8S. Ross.* Research
Dept., Ciba Pharmaceutical Products, Inc.,
Summit, N. J.

The hypotensive response of normal dogs fol-
lowing the acute administration of ganglionic
blocking agents varies with the state of anesthesia,
Unanesthetized dogs exhibit only a transient fall,
if any, in diastolic pressure, but invariably show a
reduction of systolic pressure. Dogs under barbi-
turate anesthesia exhibit marked, sustained drops
in both systolic and diastolic pressure after
ganglionic blocking agents. The transient hypo-
tension produced by acetylcholine is also po-
tentiated by barbiturate anesthesia. Gellhorn and
Redgate (Arch. int. pharmacodyn. 102: 162, 1955)
attribute this effect to depression of hypothalamic
excitability resulting in reduced vasomotor
compensatory activity. This mechanism quite
possibly is operating in the barbiturate potentia-
tion of the hypotensive effect of ganglionic block-
ing agents. In the unanesthetized dog some
vasomotor pathways may not be affected by
ganglioplegic agents and can elicit sufficient vaso-
constriction to sustain diastolic pressure. Barbi-
turates, by further reducing sympathetic activity,
may permit diastolic pressure decline. Reserpine,
which apparently reduces sympathetic outflow via
central action, can markedly lower the diastolic
pressure of the neurogenic hypertensive dog and
antagonize pressor spikes induced in these animals
by application of annoying stimuli, while gan-
glionic blocking agents antagonize the pressor
responses but generally do not markedly reduce
the diastolic level. Again it would seem that
sympathetic activity is not sufficiently reduced by
ganglionic blocking agents to effect a diastolic
depression. Antagonism of the pressor spikes
might indicate near maximal vasoconstriction in
some region which is sufficient to sustain diastolic

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

457

pressure but has not the capacity to elicit an
elevation of the existing systolic pressure.

1489. Amelioration of Myleran-induced bone
marrow damage in rats with homologous
marrow injections. R. E. MAxwELL AaNnp J. K.
Weston (introduced by A. C. Bratton, JR.)
Research Labs., Parke, Davis & Co., Detroit, Mich.
Using 20 mg/kg doses of Myleran intraperi-

toneally as a marrow-damaging agent, multiple
intravenous injections of homologous marrow into
rats within the week following the Myleran usually
induced marked amelioration of the damage
within 15 days, whether evaluated chemically or
histologically. In control animals, the mean
desoxyribonucleic acid (DNA) concentration
was 25.6+3.9 ug/mg wet weight of marrow, and
the mean nucleated cell count was 1.93+0.23 x
10° per cu mm. In Myleran-treated animals, killed
at 2 wk., the corresponding figures were 11.2+5.3
and 0.64+-0.28 X 10°. Histological examination of
sections of the femurs of this group demonstrated
marked hypocellularity with abnormal cytology.
Smear counts of 1000 cells demonstrated marked
hypocellularity, primarily in the myeloid series.
One group of Myleran-treated rats was admin-
istered 100 X 10° nucleated rat marrow cells per
animal daily for 7 consecutive days following
Myleran injection. At killing, the DNA level in
this group was 21.3+4.9. Another group of My-
leran-treated rats was given 125 X 10® nucleated
rat marrow cells per animal only on the 4th and
5th days subsequent to Myleran injection. These
animals, at killing, showed a DNA level of 21.34
3.4 and nucleated cell count of 1.30+.32 X 10°.
In both these groups, histological examination of
sections of the femurs indicated a slight hypo-
cellularity and the smear count picture resembled
that of control animals. Investigation is con-
tinuing to determine the minimal amount of
marrow and the optimal time of injection required
to result in apparent marrow normality. (This
work was done under Atomic Energy Commission
contract No. AT(11-1)-334.)

1490. Analogues of arterenol. R. S. Mc-
CuTcHEON* AND R. P. Antauist. Dept. of
Pharmacology, Med. College of Georgia, Augusta.
Three analogues of arterenol were comparatively

studied on several effector systems in dogs. The

amines were: (I) ethylnorepinephrine, (II) iso-
proterenol and (III) the N-isopropy] derivative of
ethylnorepinephrine. These amines were compared
to levarterenol and epinephrine as to potency
and effect on arterial pressure, heart rate, ECG,
intestinal motility, splenic capsule, urine output
and blood flow in various vascular beds. The
effects on the splenic capsule illustrate the general
relationships found. Epinephrine, levarterenol

458

and (I) produce primary splenic contraction; this
effect is prevented by adrenergic blockade and is
not modified by ganglionic blockade. Compounds
(II) and (III) and (I) to some extent, induce a
reflex splenic contraction by their depressor
action. This effect is prevented by either
adrenergic or ganglionic blockade. When ad-
ministered simultaneously with epinephrine, (II)
and (III) diminish the splenic contraction pro-
duced by the epinephrine. Quantitative data on
these effects will be presented. Studies on the
metabolic actions of these amines are being done.
(From the Cardiovascular Research and Training
Program supported by grants from the Natl.
Heart Inst. and American Heart Assoc.)

1491. Effects of magnesium excess and calcium
lack on frog muscle R.P. in vitro. A. R.
McIntyrrE, Paut YouNnG* AND FREDERICK
Ware.* Univ. of Nebraska College of Medicine,
Omaha, Nebr.

Sartorius muscles of Rana pipiens soaked 24 hr.
in ‘normal’ Ringer’s solution and in Ringer’s
solution containing 16 mm magnesium, showed no
significant fall in resting potential at room tem-
perature (22°C). Frog Sartorius muscle soaked in
calcium free Ringer’s suffered a fall in R.P. within
a few minutes, and in 90 min. had fallen by an
average of 10 mv. In 24 hr. the rate of R.P. fall
had markedly decreased at which time the R.P.
averaged approximately 25 mv below the controls
soaked in normal Ringer. When the muscles’
spontaneous activity in calcium free solutions
was prevented by the addition of excess mag-
nesium (20 mm) the same magnitude of fall in
R.P. was observed as in those muscles undergoing
spontaneous twitching. Hence the fall in R.P. in
calcium free solutions cannot be attributed to
contractile activity. The excess magnesium in the
concentrations used has no measurable effect on
the R.P.

1492. Effect of alloxan and ligation of the
pancreatic duct on the turnover rate of
zine-65 in the rat. Ropert J. McIsaac (in-
troduced by Doveuas S. Riaas). Dept. of
Pharmacology, Univ. of Buffalo School of Medi-
cine, Buffalo, N. Y.

We have previously found, by autoradiography,
that injected radioactive zinc-65 was retained in
high concentrations by the islets of Langerhans
of the rat pancreas for relatively long periods of
time. Some investigators have postulated that
there is a relationship between islet zine and
insulin metabolism. In order to elucidate the role
of various tissues of the pancreas in zinc metab-
olism, an attempt was made to study the turnover
of zinc-65 in the rat pancreas after one or more of
the pancreatic tissues had been destroyed. Three
groups of rats were studied: the Ist was untreated

FEDERATION PROCEEDINGS

Volume 16

and served as a control, in the 2nd group the
pancreatic duct was ligated and the acinar tissue
allowed to degenerate, and in the 3rd group the
beta cells of the islets were destroyed by alloxan,
Two rats from each group were killed at 12, 24,
48, 96 and 192 hr. and analyses of the total zine
as well as radioactive zine were made. Neither the
concentration of zinc-65 nor the concentration of
total zine of the pancreas from the diabetic
animals differed significantly from normal. On the
other hand, the total zine concentration in the
duct ligated animals was below normal. The zine-
65 concentration was less than normal for the
first 48 hr. but there was little difference between
the duct ligated pancreas and normal pancreas
from 48 to 192 hr. The results indicate that when
acinar tissue has been destroyed, the uptake of
zine-65 is decreased and the turnover rate of zine
by the pancreas is changed,

1493. Blood ammonia following administra-
tion of various hydrazino compounds.
HERBERT McKENNIS, JR. AND J. H. WEATHERBY.
Dept. of Pharmacology, Med. College of Virginia,
Richmond.

Previous investigations from this laboratory
indicate that hydrazine, isonicotinylhydrazine,
1-isonicotinyl-2-isopropylhydrazine, and _ 1-iso-
nicotinyl-2-acetylhydrazine cause fatty livers in
rabbits when administered in quantities of the
order of 0.75 mm/kg. No significant increase in fat
content was noted after a similar dose of 1, 1-di-
methylhydrazine, or after 0.115 mm/kg methyl-
hydrazine. Various investigators have reported
elevated blood ammonia values in humans suffer-
ing from certain liver disorders, particularly
alcoholic cirrhosis. The question arises as to
whether or not elevated blood ammonia is as-
sociated with liver damage following poisoning
from certain hydrazine derivatives also. Ae-
cordingly, blood ammonia values, and in some
instances spinal fluid ammonia values also, were
determined in anesthetized dogs at 2- and 5-hr.
intervals after administration of the aforemen-
tioned hydrazino compounds. In the anesthetized
(pentobarbital) but otherwise untreated dog
blood ammonia nitrogen decreases within a period
of 5 hr. from a normal value of 0.3-0.8 ug/ml to
0.1-0.2 ug/ml. Spinal fluid normally contains
little or no ammonia nitrogen. Five hr. after the
administration (i.v.) of 1.56 mm/kg hydrazine
blood ammonia nitrogen values were from 6 to
10 ug/ml and spinal fluid values 5-7 ug/ml. Of the
remaining compounds investigated only iso-
nicotinylhydrazine (1.09 mm/kg) showed a definite
tendency to cause an increase in blood ammonia
nitrogen within 5 hr.-to 1.1 ug/ml. Other com-
pounds were administered in the following quan-
tities: methylhydrazine, 0.54 mm/kg; 1,1-dimeth-

(P
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March 1956

ylhydrazine, 1.5 mm/kg; 1,2-dimethylhydrazine,
2 mM/kg; 1-isonicotinyl-2-isopropylhydrazine,
1.62 mm/kg; and 1-isonicotinyl-2acetylhydrazine
1.62 mm/kg.

1494. Cardiac rates in digitalized cats with and
without atropine. Paut L. McLain. Dept. of
Physiology and Pharmacology, Univ. of Pittsburgh
School of Medicine, Pittsburgh, Pa.

Digitoxin, 0.02 mg/kg, was administered in-
travenously to lightly etherized cats every 5 min.
until death. Eight of 20 animals received 0.2
mg/kg of atropine sulfate prior to digitalization.
Cardiac rates were counted from electrocardio-
grams recorded before and after each dose of
either drug. All animals developed typical ven-
tricular tachycardia under digitoxin. Control
rates for the 2 series were indistinguishable
(P > 0.9) as were the maximum ventricular rates
under digitoxin (P > 0.3). Minimum sinus rates
under digitoxin were the higher in the atropinized
animals, approaching significance at the 5% level.
Changes in rate were compared by analysis of
individual differences. Maximal ventricular rates
under digitoxin were higher than the correspond-
ing control sinus rates for both series (P < 0.05)
and the mean changes were statistically indis-
tinguishable (P > 0.4). However, in the atropine
group, the mean change from a post-atropine
control was not significant (P > 0.3). In fact,
the mean increase in rate due to atropine alone
was virtually identical with that attributable to
digitoxin. Minimum sinus rates under digitoxin
were significantly (P < 0.01) below the control
rates for nonatropinized but not for atropinized
animals (P > 0.3). However, in the latter group,
the differences became highly significant (P <
0.001) when postatropine rates were used as con-
trols. On this basis, there was no difference be-
tween the 2 groups. The results indicated that
atropine was without effect on the ventricular
tachycardia induced by digitoxin. As judged by
individual changes in sinus rate under digitoxin,
‘extra-vagal’ slowing was as great, on the average,
as that with the vagal mechanisms intact.

1495. 1-3-Hydroxy-N-propargyl morphinan
(Ro-1-7780), a new opiate antagonist.
RoBERT MEGIRIAN AND CHESTER W. WHITE, JR.
(introduced by Eart H. DearBorn). Dept. of
Anesthesiology, Boston City Hosp., and Dept. of
Pharmacology, Boston Univ. School of Medicine,
Boston, Mass.

The effectiveness of 1-3-hydroxy-N-propargyl
morphinan as an antagonist to alphaprodine and
to morphine was assayed in 60 experiments using
27 dogs. The drug alone had no apparent effect at
0.5 mg/kg. When it was given following doses of
2-12 mg/kg of alphaprodine or 1.5-3 mg/kg of
morphine in 8 conscious dogs, however, Ro-7780

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

459

at 0.25-0.5 mg/kg produced a rapid return of
responsiveness to normal with accompanying
improvement in ventilation and in pulse force and
rate, contrasting sharply with the controls.
Studies of ventilation in 8 dogs under pento-
barbital which received first 5 mg/kg of alpha-
prodine and then 0.25 or 0.35 mg/kg of Ro-7780
showed significant improvement in respiratory
minute volume and arterial oxygenation (P <
0.01) when the antagonist was used. When ratios
of alphaprodine to Ro-7780 of 100 or 75 to 1 were
given contemporaneously, nearly normal ventila-
tion and oxygenation of the blood were seen while
alphaprodine alone produced severe depression of
both functions. Control and test observations
were made in the same animal in all cases. Ro-7780
appeared equally effective in antagonizing mor-
phine but detailed data are not yet available. The
respiratory stimulating effect of the new drug
lasted from 30 to 45 min. in the doses used.

1496. Chlorpromazine in normal and tourni-
quet-shocked mice. R. Cart Miuiican. Natl.
Insts. of Health, Bethesda, Md.

Chlorpromazine (CPZ) therapy was studied in
tourniquet trauma in mice. Pretreatment (} hr.
before tourniquets application) prolonged survival
significantly. 12 hr. after tourniquets release
survivals were: control group, 15%; CPZ groups
(2-20 mg/kg, i.p.), 48-78%, 55 animals/group; at
21 hr. survivals were 0 and 17-30%, respectively.
CPZ pretreatment, supplemented with isotonic
NaCl (1 ml, i.p. after tourniquets release) pro-
duced increased survival. Twenty-four-hr. sur-
vivals were 17% in saline group and 54, 75 and
87% in CPZ-saline groups (2, 8 and 20 mg/kg,
respectively, 29 animals/group). CPZ was effective
with 3 and 12 hr., but not with 24-hr. pretreat-
ment, but was ineffective when administered (8
mg/kg; 1 ml saline) immediately after tourniquets
release (47 and 44% survival for CPZ-saline and
saline controls, respectively, 48 animals/group).
These data confirm Beck and Redick (Am. J.
Physiol. 183). In normal mice CPZ (20 mg/kg)
decreased rectal temperature to 29° within 1 hr.;
(this temperature remained unchanged for 12 hr.);
increased bleeding volume 20%; decreased hema-
tocrits; and reduced plasma protein levels 20-30%.
CPZ augmented the effects of intravenous fluids.
Serum and saline (1 ml volume, i.v.) given to
normal mice produced changes in bleeding volume,
hematocrit and plasma protein levels which re-
turned to normal within 2 hr.; after CPZ treat-
ment these effects were 20-30% greater and did not
return to normal within 8 hr. Chlorpromazine
sulfoxide, a metabolite, produced positive effect
on survival (pretreatment only) without affecting
rectal temperature and bleeding volume in nor-
mals.

460

1497. Effects of Pitocin, Pitressin and epi-
nephrine on the cortical activity in male
rabbits. Bruno Minz, Leo GoLpsTEIN* AND
JEANNE FuGazza.* Dept. of General Physiology,
Sorbonne, Paris.

Intravenous injection of epinephrine is regularly
followed by an increase of cortical activity meas-
ured by the mean voltage recorded per cycle and
per second. However, direct cortical application
of the same hormone is not only ineffective but
abolishes cortical reaction to subsequent in-
travenous injections (Minz aNpD GOLDSTEIN.
Federation Proc. 14: 1196, 1955). Cortical epi-
nephrine thus seems to inhibit the central action
of intravenous epinephrine. The possible reasons
for this effect have been investigated. It may be
due either to a blockage by local accumulation of
epinephrine in the brain or to an indirect action
produced by hypothalamic release of Pitressin
and Pitocin after cortical application of epi-
nephrine. (CHAMORRO AND Minz. C. R. Acad. Sci.
240: 1368, 1955). The increase in electrical activity
occurs without any change after 4 consecutive
intravenous injections of epinephrine. On the
contrary both Pitressin and Pitocin (kindly sup-
plied by Dr. Chen of Parke, Davis) produce
definite effects on the cerebral cortex. Pitressin
(1 unit i.v.) lowers the effect produced by epi-
nephrine; Pitocin (1 unit i.v.) abolishes it entirely.
An unexpected result has been obtained with
Pitocin: this hormone is capable of increasing
markedly cortical activity for periods as long as
200 sec. without any effect on blood pressure. A
mixture of 2 parts Pitressin and 1 part Pitocin
produces effects analogous to those obtained with
cortical application of epinephrine.

1498. Hypotensive activity of the combination
of a new oral adrenolytic drug and reserpine
in renal hypertensive rats. H. Minatroya
(introduced by F. P. LupuENa). Pharmacology
Section, Sterling-Winthrop Research Inst., Rens-
selaer, N. Y.

It has been shown that a new oral adrenolytic
(9-[(2-Chlorethyl)ethylaminomethy]l] anthracene
hydrochleride lowers blood pressure in renal
hypertensive rats (MrinaTtoya AND LupUENA. To
be published). The hypotensive effect of a com-
bination of the anthracene derivative and reser-
pine, in various proportions, has been compared
with the effect obtained when the drugs were ad-
ministered singly. Forty-five hypertensive rats
(male, Sprague-Dawley strain, hypertensive
2-5 months) with elevated blood pressures of
175-220 mm Hg systolic were used interchangeably,
10 rats/dose level, at intervals of 10-14 days.
Renal hypertension was produced by using a
modification of the latex-encapsulation method
(ABRAMS AND Sosin, Proc. Soc. Exper. Biol. &
Med., 64: 412, 1947). Blood pressures were deter-

FEDERATION PROCEEDINGS

Volume 15

mined in the lightly etherized animals before and
1, 2, 4-5, 6-7 and 24 hours after medication using
a microphonic manometer. The drugs, in aqueous
solution, were administered by stomach tube.
The anthracene derivative, in oral doses of 0.125,
0.5 and 2 mg/kg produced a mean maximal redue-
tion of 19.342.1, 24.941.8 and 3242.6 mm Hg,
respectively, in blood pressure in 2-4 hr. after
medication. A mean maximal reduction of 20.6+
1.9, 26.4+3.4 and 38.34+2.4 mm Hg in blood pres-
sure was obtained with oral doses of 0.5, 1 and 2
mg/kg, respectively, of reserpine in 5-6 hr. When
0.125 mg/kg of the anthracene derivative and 0.5
mg/kg of reserpine was combined, a marked
potentiation in the hypotensive activity was ob-
tained. A mean maximal reduction of 35.6+1.5
mm Hg in blood pressure was obtained in 4-6 hr.
A similar potentiation in hypotensive effect was
obtained by administering 0.5 and 2 mg/kg of the
anthracene derivative and 0.5 mg/kg of reserpine.
A mean maximal reduction in blood pressure was
41.54+2.2 and 46.2+4.5 mm Hg, respectively. The
degree of potentiation was not altered by increas-
ing the dose of reserpine. The duration of hypo-
tensive activity was more than 24 hr.

1499. Dose-response of epinephrine and of
procaine amide during hypothermia.
SaMvUEL Q. MitTcHELL,* Frances Da Cosrta,*
Water M. Booker, Ropert R. WuHEATON*
AND Ropert J. Rosprnson. Dept. of Pharma-
cology, Howard Univ. Med. School, Washington,
D.C.

The employment of hypothermia as an adjunct
to anesthesia in cardiac surgery has given rise to
important questions regarding the effectiveness or
lack of effectiveness of certain cardiac drugs dur-
ing the state of hypothermia. We regarded it both
interesting and important to study the dose-
response of epinephrine, as well as_procaine-
amide before and during hypothermia. Dogs
were used throughout these studies. The animals
were first anesthetized with pentobarbital sodium
(30 mg/kg). Control electro-cardiograms were
taken, then the animals were administered epi-
nephrine in doses of 0.01, 0.05, 0.1, and 0.15 mg/kg;
or in other experiments the animals were ad-
ministered procaine-amide in doses of 1, 5, 10, 15,
20 mg/kg. Electro-cardiograms were run im-
mediately after each administration and blood
samples were taken for sodium and potassium
determination. Hypothermia was achieved by
using a bath tub of ice water and subsequently ice
packs. The epinephrine doses (or the procaine-
amide) were then repeated as before hypothermia.
Electro-cardiograms were run and blood samples
were drawn as before hypothermia. The results so
far have indicated that at normal body tempera-
ture, a dosage of 0.1 to .15 mg/kg, epinephrine
causes a tremendous increase in rate, with ex-

1-n

the

stil

lat

lume 1

ore and
n using
\queous
1 tube.
f 0.125,
| redue-
im Hg,
r. after
fF 20.64
d pres-
1 and 2
. When
and 0.5
marked
vas ob-
9.61.5
4-6 hr.
ct was
x of the
erpine,
ire was
ly. The
ncreas-
f hypo-

ind of
ermia.
SOsTA,*
EATON*
-harma-
ington,

idjunet
rise to
ness or
gs dur-
it both
» dose-
ocaine-

Dogs
ininials
sodium
s were
ed epi-
mg/kg;
‘re ad-
10, 15,
in im-
blood
assium
red by
itly ice
ocaine-
1ermia.
amples
sults so
mpera-
»phrine
ith ex-

March 1956

trasystoles‘and ventricular preponderance. These
doses of epinephrine during hypothermia, on the
other hand, caused no increase in rate nor any
suggestion of irritability of the muscle. The QRS
complexes did not become biphasic, nor did the T
waves show significant alterations in height or
directions. An attempt to increase the dose during
hypothermia beyond .15 mg/kg resulted in a
gradual production of low voltage in all leads and
a steady development of an irresponsive heart.
Procaine-amide at normal body temperature
caused chiefly an elevation of the T wave in
leads 2 and 3 when doses of 10 mg/kg or greater
were used. During hypothermia this dose caused
a marked heightening and elongation of the T
wave, and the subsequent cessation of the heart
beat. The results will be reported in correlation
with changes in serum potassium and sodium.

1500. New hypotensives with central and
peripheral actions. RoBert L. Morritt* anp
Rosert K. 8. Lim. The Miles-Ames Research
Lab., Elkhart, Ind.

Ninety-two new piperazine derivatives have
been evaluated for hypotensive action in unanes-
thetized dogs rendered hypertensive by sino-aortic
exclusion. The most active of these, 1-[2-(3,4-di-
methoxypheny]l)-1-methylethy]]-4-furfury] - piper-
azine and 1-isobutyl-4-[2-(3,4-dihydroxy phenyl) -
1-methylethyl]-piperazine, exhibited sustained
hypotension when given either intravenously
or orally at 5% of the ipso or less. These com-
pounds blocked the blood pressure responses
to noradrenaline, adrenaline and _ splanchnic
nerve stimulation, but not those to acetylcholine
or cardiac vagus stimulation. Transmission
through the superior cervical ganglion was not
impaired. The compounds were studied in dogs
in which the head circulation was isolated from
that of the trunk, but the neural connections
(spinal and vagal) were intact, the circulation in
the vasoisolated head being maintained by a donor
dog or a finger pump perfusion system. Introduc-
tion of the compounds into the circulation of the
vaso-isolated head induced sustained hypotension
in the vaso-isolated trunk as well as in the donor,
while intravenous injection into the vaso-isolated
trunk resulted in a sustained hypotension in the
trunk alone. The blood pressure response to
stimulation of the vasomotor center in the medulla
was blocked by direct introduction of micro-
amounts of the compounds (through the stimu-
lating electrode) into the area stimulated. Thus,
the hypotension is produced partly by blockade
of the vasomotor center in the medulla and partly
by blockade of adrenergic terminations at the
periphery. Chance alternation of the neurons
blocked centrally and peripherally will permit
greater hypotension with fewer side effects at a

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

461

lower dose than if the blockade were wholly at one
site.

1501. Influence of stimulation frequency on
the transmission through parasympathetic
and sympathetic ganglia under various
pharmacological conditions. R. Moors,*
E. Stae* anp J. A. ScHNEIDER. Research Dept.,
Ciba Pharmaceutical Products Inc., Summit,
N.J.

It is known that the rate of stimulation in-
fluences form and size of the postsynaptic sympa-
thetic potentials in the cat. Since similar data
are not available for parasympathetic ganglia it
was attempted, first, to investigate the effect of
repetitive stimulation on the transmission through
the ciliary ganglion before and after injection of
various blocking agents; and secondly, to com-
pare the responses with those obtained from
superior cervical ganglia. Preganglionic fibers of
the cat’s superior cervical and ciliary ganglion
were stimulated with maximal pulses of constant
parameters, the stimulation frequency being the
only variable. During trains of preganglionic
volleys from 0.1 to 10 or 15 pps the amplitude of
the postganglionic potentials was maintained
or even slightly increased. At frequencies above
15 pps the amplitude diminished rapidly to reach
eventually one-fifth or less of the original height
at 40 pps, the response of the sympathetic ganglion
being more readily depressed than the response of
the parasympathetic. The study of parasympa-
thetic postsynaptic potentials through a wide
range of stimulation frequencies offers the possi-
bility not only to quantitate the effectiveness of
pharmacologically induced ganglionic blockade
but also to detect possible selective blocking
effects on parasympathetic and sympathetic
ganglia. Thus, for instance, it was demonstrated
that chlorisondamine chloride (Ecolid T. M.)
blocks transmission through the ciliary ganglion
at low stimulation frequencies which do not affect
the superior cervical ganglion at all, a finding
which might explain the blurred vision often
observed during the treatment of hypertensive
patients with this drug.

1502. High spina! anesthesia as an adjunct to
hypothermia in man. GrorGE MORALES AND
Apo MorELLo (introduced by Louts R. Orx1n).
Dept. of Anesthesiology, Albert Einstein College
of Medicine, Yeshiva Univ. and Dept. of Neuro-
surgery, New York Univ. Post-Grad. Med. School.
Seven patients at Central Islip State Hospital

underwent 42 trials of hypothermia at weekly

intervals as a type of shock therapy for schizo-
phrenia. In 38 of the 42 trials, cooling was ac-
complished with refrigerating blankets. The re-
maining 4 studies were duplicated without cooling.
The anesthetic technics were divided into 3

462

groups: I, 10 trials with thiopental. II, 12 with
relaxants and thiopental. III, 20 with high spinal
anesthesia and thiopental. The rate of cooling per
degree Farenheit in each group was I, 20-25 min.,
II, 15 min., and III, 10 min. The most important
influence upon the rate of cooling was the amount
of shivering but the effect of spinal anesthesia on
the peripheral circulation cannot be ignored.
The oxygen uptake is presented as change from
the B.M.R. Its tendency was to follow the tem-
perature. Marked deviations occurred with shiver-
ing or hypotensive episodes. The changes observed
in the ECG in the order of their appearance
with decreasing temperature were increased
voltage of T waves, prolonged P-R interval,
elevated ST segment, elevation and notching
of R wave, disappearance of P waves and oc-
casional P.V.C.’s. Bradycardia appezred early in
patients with spinal anesthesia. These changes
started at 90°F; all were reversible with warming.
Changes occurred at higher temperatures in suc-
cessive coolings on the same subject. The renal
plasma flow and glomerular filtration were de-
creased by 30-50%. Tubular absorption was also
decreased.

1503. Cardiovascular actions of NHI-196, an
alkaloid from the seeds of Ormosia pana-
mensis, a Central American tree. NEIL C.
Moran, GERTRUDE P. QUINN AND WILLIAM M.
Butter, Jr. (introduced by BERNARD B.
Bropie). Lab. of Chemical Pharmacology, Natl.
Heart Inst., Bethesda, Md.

NHI-196, a quaternary-like base derived from
the seeds of Ormosia panamensis, produces a long-
lasting fall in arterial pressure in dogs and cats.
The minimal effective i.v. dose is from 10 to 100
ug/kg in dogs. The vasodepressor response on i.v.
administration is characterized by a latent period
of 30-120 sec., a slow fall in pressure, tachycardia
with high doses, cutaneous flush, hemoconcentra-
tion and tachyphylaxis. With doses up to 1 mg/kg
it produces no adrenergic or ganglionic blockade,
is not blocked by atropine, is active in the de-
buffered dog, does not depress the heart, produces
no consistent direct vasodilatation on femoral
artery injection, does not release histamine into
the blood, is active in the eviscerated dog, and is
not blocked by pyrilamine but is by convulsant
doses of tripelennamine. NHI-196 is depressor in
only 25% of cats with high spinal cord transection
and is uniformly active in decerebrate cats.
However, cervical cord section or cord destruc-
tion in dogs does not abolish the action. The
latent period on intracarotid artery injection is
12-40 sec. in contrast to the longer latent period on
i.v. administration. Other actions include pulmo-
nary artery hypertension and increased intra-
tracheal pressure, stimulation of intestinal

FEDERATION PROCEEDINGS

Volume

motility, piloerection, slight sedatior and barbi.
turate potentiation in dogs but not ia rats, oe
casional cutaneous edema of the snout and eyelids
in dogs and vasopressor responses in rabbits. The
present evidence suggests a predominantly periph-
eral site of vasodepressor action in dogs, possibly
an atypical histaminergic effect, with a possible
associated vasomotor center depression. (NHI-
196 provided by Drs. Helen A. Lloyd and Evan (C,
Horning of the Lab. of Chemistry of Natural
Products, Nat’l. Heart Inst.)

1504. Metabolic conversion of ethyl p-nitro-
phenyl thionobenzene phosphonate (EPN)
to an anticholinesterase agent. SHELDON D,
Murpuy* aNd KEenneETH P. DuBots. Dept. of
Pharmacology, Univ. of Chicago, Chicago, Ill.

A study of the metabolism of ethyl p-nitro-
phenyl thionobenzene phosphonate (EPN) was
undertaken to extend the available information
on the mechanism of action of this compound. A
purified sample of EPN produced no inhibition
of brain cholinesterase in vitro at a final concen-
tration of 1 X 10° m. However, after incubation
of the compound with liver slices of Krebs-Ringer-
phosphate buffer under aerobic conditions a
metabolite with strong anticholinesterase activity
was formed. The conversion was demonstrated by
incubation of 1 X 10-* m EPN with liver slices in
Warburg vessels using oxygen as the gas phase.
The anticholinesterase activity of the incubation
mixture and the relative concentration of the
active metabolite were estimated using brain
cholinesterase measurements as an assay procedure
according to the method developed previously in
this laboratory (J. Pharmacol. & Exper. Therap.
99: 376, 1950). After incubation of EPN with 15
to 25 mg of liver slices (d. wt.) for 60 min. a con-
centration of 1 X 10-'m with respect to the original
compound produced 50% to 75% inhibition of
brain cholinesterase in contrast to the absence of
inhibition by this quantity of unincubated EPN.
Addition of 8-diethylaminoethyl diphenylpropy]-
acetate hydrochloride (SKF 525A) to the medium
in which the EPN was incubated with liver slices
markedly inhibited the conversion to an anti-
cholinesterase agent. The presence of 1 X 10%
M SKF 525A caused 58% inhibition of the meta-
bolic conversion of EPN.

1505. Absorption and excretion of 5-methyl-5-
phenylhydantoin in the rat. Joe B. Nas#
(introduced by GEorcEe A. Emerson). Dept. of
Pharmacology and Toxicology, Univ. of Texas
Med. Branch, Galveston.

After oral administration of 35 mg/kg of
5-methyl-5-phenylhydantoin-5-C™ to fasted rats,
concentrations of radioactivity in blood reached a
mean peak level in 3 hr. Radioactivity in the urine

91-

pr
5-n

pr
ch

res

olume 1%

id barbi-
rats, 0¢
d eyelids
yits. The
y periph.
possibly
possible
. (NHI
Evan (,
Natural

)-nitro-
> (EPN)
LDON D,
Dept. of
Til.
p-nitro-
N) was
rmation
ound. A
hibition
concen-
ubation
Ringer-
tions a
activity
ated by
slices in
phase,
ubation
of the
+ brain
cedure
usly in
Therap.
with 15
a con-
riginal
tion of
ence of
1 EPN.
propyl-
nedium
r slices
1 anti-
x 10°
-meta-

hyl-5-
NASH
lept. of
Texas

kg of
1 rats,
ched a
2 urine

March 1956

of treated’animals could be demonstrated within
9 min. after dosing. Urinary excretion accounted
for approximately 93% of the dose administered.
About 70% were eliminated the first 24 hr. Mean
quantities of 19.3% and 3.2% were excreted on the
dnd and 3rd days. Fecal excretion at 72 hr. com-
prised from 3.4-5.5% of the administered dose.
Expired air collected for 8} hr. contained no
measurable CO». Urine was extracted with ether
to recover the radioactive material. Paper chro-
matographic techniques, microsublimation and
isotope-dilution methods were employed to
ascertain the nature of the substance. From
91-93% of the total radioactive urinary excretion
products could be characterized as unchanged
§-methyl-5-phenylhydantoin.

1506. Mechanism of the prolonged adrenergic
blocking action of Dibenzyline. Marx
Nickerson. Dept. of Physiology and Med. Re-
search, Univ. of Manitoba Faculty of Medicine,
Winnipeg, Canada.

Although several lines of indirect evidence
suggest that the persistent adrenergic blockade
produced by 6-haloalkylamines is due to stable
chemical binding of drug in blocked tissues, it also
has been claimed that prolonged blockade may
result from accumulation of active drug in and its
slow release from fat depots (AXELROD, ARONOW
AND Brop1k, 1952). In the present experiments the
roles of these 2 possible mechanisms have been
investigated in vitro and in vivo. Surviving strips
of rabbit aorta suspended in oxygenated Krebs-
Ringer solution were exposed to Dibenzyline for
5 min. and the blockade of responses to adrenaline
followed for 72-96 hr. Responses were reduced to
<5% of the control values immediately following
treatment and recovered only slightly during the
period of study. In contrast, responses of strips
run parallel to the above but blocked daily with a
short-acting blocking agent (piperoxan) were
still >50% of the control values at the end of the
experimental period. These results indicate that
Dibenzyline blockade is well sustained in vitro
in the absence of any ‘depot’ of active drug.
In intact rats prior administration of thiosulfate,
which chemically inactivates Dibenzyline, pre-
vented blockade. However, thiosulfate did not
alter the established blockade, indicating that
continued release of active drug is not necessary
for prolonged action. That some active drug enters
the circulation long after intramuscular ad-
ministration is indicated by the fact that if the
blood thiosulfate level declines as late as 10 hr.
after injection, significant blockade can develop.

1507. Hexosamine concentration in normal
and atheromatous human aortic con-
nective tissue. Nancy L. Nose, Rosert J.
Boucex, Kune-Yine T. Kao anp HEtLen C.

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

463

ParTIN (introduced by Ratrpu JONES, JR.).
Miami Heart Inst., Univ. of Miami School of
Medicine, Miami, Fla.

Intimal connective tissue was dissected from
human aortas obtained at necropsy and divided
grossly into 3 areas: normal, early atheroma and
advanced atheroma. These tissues were first
homogenized in saline to separate the soluble
ground substance from the insoluble scleroprotein
residue, which was subsequently incubated with
testicular hyaluronidase and the resultant residue
was hydrolyzed with hydrochloric acid. Hexosa-
mine concentration of these fractions was deter-
mined. With progressive atheromatosis, there is a
decrease in the concentration of saline-soluble
hexosamine and an increase in hyaluronidase-
susceptible hexosamine. There seems to be no
sexual difference between the concentrations of
saline-soluble hexosamine in the connective tissue
of the 3 aortic areas. However, it appears that
normal female aortic connective tissue has a lower
concentration of hyaluronidase-susceptible hex-
osamine than normal male tissue. After gross
atherosclerotic changes begin, there is no apparent
sexual difference between the hexosamine con-
centrations of this fraction. No apparent relation-
ship appears to exist between the age of the
individual and the hexosamine concentration of
human aortic connective tissue. The observed
differences in hexosamine concentration of the
connective tissue of the three aortic areas will be
related to the lipid and protein changes which
occur with developing atherosclerosis. (Sup-
ported by Research Grant H-2087 Natl. Insts. of
Health, PHS)

1508. Evaluation of drugs affecting the psyche
by cat behavior studies. Stata NorTON AND
E. J. pr Beer. Wellcome Research Labs., Tuckahoe,
Ni ¥.

A method was set up to test drugs affecting
the psyche using spontaneous behavior patterns
in cats. Large numbers of cats were observed to
determine what behavior patterns could be ob-
served under laboratory conditions. No deliberate
conditioning of responses was attempted. Four
behavior patterns were selected for study: 1) so-
ciability; 2) contentment; 3) excitability; and 4)
hostility. In a general way, categories 1 and 4 were
selected to represent opposite reactions of the
animals directed toward the observer, and cate-
gories 2 and $ to represent opposite patterns re-
flecting the emotional attitude of the cat to all
of his accustomed surroundings. Each behavior
pattern was defined by 5 components which
were found to be associated in control studies.
The components were selected to provide un-
equivocal all-or-none behavior so that the data
could be quantitated. A scoring system was set

464

up based on the frequency of occurrence of the
components of the patterns. The least common
components were considered to impart the most
information about the cat and were therefore
given the highest scores. Using this technique
several drugs were tried, including chlorpromazine,
pentobarbital, rauwolfia and morphine. All of the
drugs modified some of the behavior patterns
but in different ways and to a greater or lesser
extent. All compounds were given orally. Doses of
lactose were used as controls and produced no
significant changes in the behavior patterns.

1509. Effect of secretin and mecholyl on the
excretion of urinary lipase in man. MARTIN
M. NotruMaNn, JosepH H. Pratt anp ALLAN D.
Cattow.* New England Ctr. Hosp. and Tufis
Univ. Med. School, Boston, Mass.

We have previously demonstrated that a fat-
splitting enzyme is present in the urine of dogs
and that the enzyme is identical with pancreatic
lipase. The fat-splitting enzyme has since been
detected regularly in the urine of man. In healthy
individuals its concentration ranges from 0.1 to
0.75 units, that means the values in the urine and
in the serum are about the same. Values of 1.0
unit and more are considered to be significant
increases. The elevated figures ranging from 1.0
to 3.35 units have been found in pancreatic disease.
In pancreatic carcinoma, however, the urinary
lipase has been less than 1 unit, at a time when the
other pancreatic enzymes in serum and urine
have also been found normal. Healthy individuals
have been given secretin or mecholyl and each
urine specimen for 24 hr. has been tested for
urinary lipase. The injection of secretin or mecholy]
causes a rise of urinary lipase well beyond 1.2
units. In pancreatic carcinoma the response is
different. After injection of tha drugs there is a
decrease in the concentration of lipase in the
urine which in some specimens may amount even
to a complete disappearance of the enzyme.
‘The response has been the same whether the tumor
has been localized in the head or in the body or
tail of the organ. The clinical implications of these
findings are obvious. (Supported by grants from
the American Cancer Society and the Damon
Runyon Fund).

1510. Progesterone and alpha tocopherol in
experimental epinephrine-thyroxine
teriosclerosis and in cholesterol-induced
atherosclerosis. Y. T. OxrstTerR, Oscar F.
Davis* AND BERNARD FRrIEDMAN.* Dept. of
Pharmacology and Exptl. Therapeutics and the
Grad. School, Stritch School of Medicine of Loyola
Univ., Chicago, Ill.

A high incidence (89.5%) of a severe degenera-
tive aortic sclerosis, essentially of the tunica

ar-

FEDERATION PROCEEDINGS

Volume |

media, is produced in rabbits by using epinephring
and thyroxine following the method previous}
reported by us. The lesions are rapidly induced ip
15 days or less. Similarly, an essentially intima]
atherosclerosis is induced in rabbits treated for}
days or less with intravenous and subcutaneoy
injections of cholesterol suspensions. Alphy
tocopherol was studied in these scleroses because
reports from Canadian and European sources hay
indicated that alpha tocopherol produces im.
provement in clinical atherosclerosis. The report
of low beta lipoprotein blood levels in young
females, as well as low 8.F. 10-30 Gofman fraction
in pregnancy led to the investigation of the effects
of progesterone on these scleroses. Five different
groups of rabbits, 8-19 per group, were given
the epinephrine-thyroxine regimen which pro-
duces the medial arteriosclerosis. In addition, each
group received 1 of the following agents by sub-
cutaneous injection: 1) progesterone—50 mg/day;
2) progesterone—75 mg/day; 3) alpha tocopherol—
100 mg/day; 4) alpha tocopherol—200 mg/day;
5) alpha tocopherol—600 mg/day. None of these
groups of animals demonstrated any significant
differences in incidence or severity of the induced
medial sclerosis when compared to the conti
group of epinephrine-thyroxine alone. Three
different groups of rabbits, 6-14 per group, were
given the cholesterol regimen which produces the
intimal atherosclerosis. In addition, each group
received 1 of the following agents by subcutaneous
injection: 1) progesterone—75 mg/day; 2) alpha
tocopherol—200 mg/day; 3) alpha tocopherol—
600 mg/day. The group of animals receiving 7%
mg/day of progesterone exhibited a much lower
incidence of the intimal aortic sclerosis than the
other 2 groups or the control group receiving only
cholesterol. The incidence in the progesterone
group was 46%, 6 animals in a total of 13 animals,
while the cuntrol group had an incidence of 71.8%
23 animals in a total of 32 animals.

1511. Simultaneous radioassay of compounds
containing tritium and carbon-14 using 4
liquid scintillation counter. Grorce T.
Oxita, Jon J. KABARA AND GEorGE V. LERorY
(introduced by E. M. K. Geriina). Argonne
Cancer Research Hosp. and Dept. of Pharma
cology, Univ. of Chicago, Chicago, Ill.

The feasibility of counting double labeled
compounds containing tritium and C™ by the
liquid scintillation counting method was in-
vestigated. A Tri-Carb liquid scintillation spec-
trometer (Packard Inst. Co., La Grange, IIL)
with a 2 channel pulse height analyzer was em-
ployed for assaying a series of samples containing
mixtures of known activity of the 2 isotopes.
By taking advantage of the differences in the
energy spectrum of tritium and C", it is possible

Volume |

yinephring
yreviousl
nduced in
ly intimal
ited for %
cutaneous
s. Alphy
s because
irces haye
luces im.
ne reports
in youn
fraction
she effects
| different
are givel
rich pro-
tion, each
s by sub-
) mg/day;
opherol—
mg/day;
of these
ignificant
e induced
e conti
>. Three
yup, were
duces the
ch group
utaneous
2) alpha
»pherol—
eiving 75
ich lower
than the
ving only
yesterone
animals,
of 71.8%

apounds
using a
ORGE T,
'. LeRoy
Argonne
Pharma-

labeled
/ by the
was in-
on spec:
ige, Ill.)
was em-
ntaining
isotopes.
s in the
possible

March 1956

to determine the amount of radioactivity for
each isotope. Initially, a simultaneous equation
was employed to calculate the radioactivity con-
tributed by each of the isotopes. However, this
method proved to be impractical due to the wide
range of errors found in the assay. Therefore, 2
other counting methods were investigated. One
method (‘screening’ method) required counting
both isotopes simultaneously at a given dis-
criminator range and then counting the pulses due
to C' alone at a higher discriminator range and
lower photomultiplier tube voltage in order to
screen out all tritium pulses. The other method
(‘discriminator-ratio’ method) involves the use of
the ratio of the counts per minute (from standard
sample containing the individual isotope) ob-
tained from the 2 channels at the appropriate
discriminator and tube voltage setting. A mathe-
matical equation is then derived which utilizes
the discriminator ratio for each isotope instead of
the counting efficiency as used in the 2 previous
methods. Of the 3 counting methods investigated
the ‘discriminator-ratio’ method proved to be
the most suitable. The standard error of the
radioassay for both isotopes by this method was
less than +5%. The range of radioactivity em-
ployed was between 200-1200 dpm for C* and
900-9000 dpm for tritium.

1512. Protection against acute hydrazine
death in mice. Joun F. O’LEaArRy ANpD Arti H.
OIKEMUuS (introduced by J. H. Writs). Pharma-
cology Branch, Med. Labs., Army Chemical Ctr.,
Md.

Mice acutely dosed with hydrazine die either in
convulsions or in postconvulsive depression.
Various CNS depressant compounds including
hypnotics, lissives and anticonvulsants were
tested in mice for ability to reduce mortality due
to a certainly lethal dose of hydrazine (LDg9).
The various depressant compounds were given at
such times prior to hydrazine as to insure full
intensity of action at the time of hydrazine injec-
tion. All depressants were given in maximal non-
lethal doses (0.2 X LDso, i.p.). Each compound
was tested in a group of at least 10 mice. Results
showed that mesantoin, phenobarbital and
3-(3-methyl-1-pentynyl) carbamate were able to
prevent hydrazine deaths in significant propor-
tions, but all others of the compounds tested were
completely ineffective. Certain structural char-
acteristics are common to the effective com-
pounds, but structure-activity experiments are
not complete. Possible factors other than structure
are duration of action, composite pharmacologic
interaction with hydrazine effects and distribu-
tion and fate in the body.

1513. Isolation of an elastase fraction from
malt diastase. Irving ONESON, FRANCES

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

465

Decisus, GEOFFERY LoRD AND ALFRED BLOCH
(introduced by Watton Van WINKLE). Ethicon,
Inc., New Brunswick, N. J.

A fraction high in elastase activity has been
isolated from commercial samples of malt diastase
by a method essentially similar to that of Hall
(Biochem. J. 59: 459, 1955) for pancreatic elastase.
Activity was measured towards a nonheat treated
substrate of bovine ligamentum nuchae shown to
consist of 83% elastin and 17% collagen. The
elastase was found to be inert towards collagen.
Elastin substrates from which collagen had been
removed by the drastic methods common in the
literature showed a greatly increased suscepti-
bility towards elastase. Malt elastase had a pH
optimum of 8.5-8.7 in 0.1 m sodium borate buffer
and was strongly inhibited by a number of salts
including sodium chloride. The latter was shown
to also inhibit pancreatic elastase. Glycine and
tris buffers showed no inhibition towards elastase.
Optimum temperature for activity was at 45°C
over a half-hour period. Inactivation occurred at
60°C. Activity was initially independent of sub-
strate concentration. Histological studies of
elastin containing tissues treated with extracts
of malt diastase supported the contention that an
elastase was involved.

1514. Hypnotic potency of thiopental sodium
and thiamylal sodium in man. Louis R.
ORKIN AND GEORGE Moratsgs.* Dept. of Anesthe-
siology, Albert Einstein College of Medicine,
Yeshiva Univ., New York City.

A previous communication (Federation Proc. 13:
393, 1954) indicated that the hypnotic potency of a
drug may be quantitated by the reduction in the
amount of thiopental necessary to produce un-
consciousness in man. This phase of the investiga-
tion is concerned with the variation between
individuals and the daily variation within the
same individual. Thiopental (25 mg/ml) was
administered to each of 10 patients on 3 successive
mornings. Ten other patients were utilized as
subjects for the same regimen employing thio-
pental in concentration of 10 mg/ml and 5 addi-
tional patients received thiamylal 25 mg/ml. All
patients received 1 ml every 30 sec. until they
failed to respond to the command ‘Open your
eyes!’ The 95% confidence limits of the mean of 3
determinations in the same individual is +30 mg.
The 95% confidence limits of a single determina-
tion in 2 different individuals is +426 mg. The
individual response to barbiturate hypnosis was
not related to age, sex, weight or basal metabolic
rate of the patients studied. The data to be
presented will indicate that under similar cir-
cumstances: 1) an individual will require the same
dose of barbiturate to reach the same degree of
hypnosis. 2) Pifferent individuals vary widely in

466

the dose-response relationship. 3) This variation
is not related to the known factors often employed
as base lines for drug administration.

1515. Headache: reactivity of bulbar conjunc-
tival vessels during the migraine type of
headache and muscle contraction head-
ache. ApriAN M. OstTFELp* anp Haro.p G.
Wo rr. Depts. of Medicine (Neurology) and Psy-
chiatry, New York Hosp.-Cornell Med. Ctr., New
York City.

Slit lamp examination of bulbar conjunctival
vessels was performed in 48 headache subjects and
14 controls on approximately 300 occasions and
over 400 photographs were made. a) During mi-
graine headache there predictably occurred,
largely on the side of headache, dilatation of arte-
rioles and venules, increased numbers of patent
capillaries, conjunctival edema and local burning
pain. On topical application of serial dilutions of
isotonic buffered solutions, arteriolar and capil-
lary sensitivity to levarterenol decreased and sen-
sitivity to acetylcholine increased. Topical corti-
sone increased sensitivity to levarterenol. After
intravenous ergotamine tartrate or levarterenol
vasodilatation, pain and edema promptly termi-
nated and the bulbar conjunctival vessels returned
to the premorbid state. During migraine attacks,
the predictable occurrence of large and minute
vessel dilatation, pain and lowered deep pain
thresholds and local edema support the inference
that minute vessel dilatation permits leakage into
tissue locally of a substance which lowers pain
thresholds and enhances pain due to artery dilata-
tion. b) During muscle contraction headache there
were predictably bilateral arteriolar constriction,
fewer visible capillaries, decreased reactivity to
acetylcholine and increased response to levar-
terenol. This vascular pattern was reproduced by
intravenous levarterenol, was unaffected by hexa-
methonium blockade and after cervical sympa-
thectomy. Such headache was transiently dimin-
ished by intravenous regitine and by amy] nitrite
inhalation. The predictable occurrence during
muscle contraction headache of bulbar conjunc-
tival ischemia concurrently with large vessel
constriction supports the thesis that ischemia
of contiguous skeletal muscle is relevant to pain
mechanisms during sustained skeletal muscle
contraction about the head.

1516. Use of ion exchange resins in removing
exogenous toxins. ARTHUR J. PALLoTTA* AND
THEODORE Koppanyi. Hazleton Labs., Falls
Church, Va., and Georgetown Univ. Med. School,
Washington, D.C.

Design of a new apparatus of simple construc-
tion for ease in operation was described. It consists

FEDERATION PROCEEDINGS

Volume 1§

essentially of a flow meter, a pliable plastic column
with screw couplings on either end for dismantling
and an air and clot catcher. Further studies indi-
cate that not only barbiturates can be removed by
this method but also salicylates and bromides,
Preliminary studies in dogs indicate that as much
as two-thirds of the circulating bromide can be
removed within $ hr. Comparable results have algo
been obtained with salicylates. No toxicity hag
been observed to date other than the removal of
white cells and platelets. Preliminary study indi-
cates this removal may be by mechanical trapping
since simple manipulation of the plastic bag causes
a release of these cells.

1517. Influence of histamine and histamine
liberator, compound 48/80, on blood glucose
of unanesthetized animals. C. A. PAapacos-
Tas,* P. MozpENn* AND Ear. R. Loew. Dept. of
Physiology, Boston Univ. School of Medicine,
Boston, Mass.

Histamine injected into the adrenal artery or
intravenously in several species of animals ig
known to liberate epinephrine as judged by pressor
responses, retraction of the nictitating membrane
and hyperglycemia. Hyperglycemia occurs during
anaphylactic and peptone shock and could be due
to direct stimulation of the adrenal medulla by
liberated histamine or to reflex sympathetic ac-
tivity coincident with severe shock. Experiments
were therefore made in which graded doses of his-
tamine, or histamine-liberator 48/80, were injected
intravenously in dogs and rabbits to determine
whether hyperglycemia could be demonstrated in
the absence of moderate or severe grades of shock.
A significant rise in blood glucose of about 15 mg %
occurred in dogs and rabbits only when the dose
of histamine base was increased to 200 me/kg, an
amount sufficient to induce moderate or severe
symptoms in the animals. This finding suggests
that the hyperglycemia induced with histamine is
related to reflex sympathetic activity induced by
the shock-like state. A relatively high dose of
48/80 (0.5 mg/kg) in 6 rabbits produced no increase
in blood glucose except in 2 animals which mani-
fested toxic symptoms. In 7 dogs the same dose of
48/80 produced prostration in 5 min., a significant
fall in blood glucose of 14 mg % in 10-20 min. and
an increase of 11 mg % above normal in 60 min.
Thus, 48/80 exerts slight, transient hypoglycemic
activity in dogs at a time when histamine and
anaphylactic shock produce hyperglycemia. This
hypoglycemic action of 48/80 may not be related
to histamine release. (Supported by a grant from
Parke, Davis & Co., Detroit.)

1518. Effects of fractionated whole-body x-ir-
radiation on phosphatase activity. DoNALD
F. Perersen* anp Kennetu P. DuBors. U.S.

olume 1§

e¢ column
mantling
dies indi-
noved by
yromides,
as much
e can be
have also
icity hag
moval of
udy indi-
trapping
ag Causes

stamine
| glucose
PAPACOS-
. Dept. of
Uedicine,

artery or
1imals ig
y pressor
em brane
rs during
id be due
dulla by
hetic ac-
eriments
es of his-
: injected
etermine
trated in
of shock,
15 mg %
the dose
ie/kg, an
ir severe
suggests
amine is
Juced by

dose of
) increase
ch mani-
e dose of
gnificant
min. and
. 60 min,
glycemic¢
nine and
iia. This
e related
ant from

dy x-ir-
DonaLD
us. US.

March 1956

Air Forge Radiation Lab. and Dept. of Pharmacol-

ogy, Univ. of Chicago, Chicago, Ill.

Radiation-induced increases in adenosine tri-
phosphatase and 5-nucleotidase activity of the
spleens and thymus glands of rats were observed
in this laboratory (Am. J. Physiol. 176: 282, 1954)
following exposure to single sublethal and lethal
doses of 250 KVP x-rays. In the present study
cumulative injury to the hematopoietic organs of
rats exposed repeatedly to doses of x-irradiation
ranging from 10 r to100 r at intervals ranging from
6 to 48 hr. was measured by periodic adenosine
triphosphatase and 5-nucleotidase assays. These
measurements indicated that daily exposures to
50 r and 100 r caused maximum increases in the
spleen phosphatase activity within 5-7 days after
which time reversal toward normal levels occurred
in spite of continued exposure. Shortening the re-
covery period between exposures from 24 hr. to 12
hr. after 50 r and 100 r resulted in persistent in-
creases in enzyme activity. Doses of 10 r and 25 r
administered daily caused smaller increases in
adenosine triphosphatase and 5-nucleotidase ac-
tivity suggesting temporary equilibrium between
radiation injury and repair processes. Doses of
less than 50 r/day caused no significant changes in
phosphatase activity of the thymus gland indi-
cating a differential sensitivity between the spleen
and thymus to repeated whole-body irradiation.
The median mortality occurred after the accu-
mulation of 2725r at the rate of 100 r/day and after
4050 r at 25 r 4 times/day. These data indicate that
both the magnitude of the dose and frequency of
exposure markedly influence the capacity of hema-
topoietic tissues to hydrolyze phosphate esters.

1519. C-isotope effect on the ion exchange
chromatography of specifically labeled
amino acids. K. A. Prez* anp H. Eaaue. Natl.
Inst. of Dental Research and Natl. Microbiological
Inst., Bethesda, Md.

A number of amino acids labeled in various posi-
tions with C were chromatographed on a 100 x
0.9 em column of Dowex 50, using pH gradient elu-
tion with citrate buffer. With 1-C- or 2-C'-la-
beled compounds, the amino acid peak in the efflu-
ent fractions as determined by C" activity did not
coincide with that found by ninhydrin color, but
followed closely. In consequence, the specific ac-
tivity of successive fractions within a single peak
increased continuously. The logarithm of the spe-
cific activity increased linearly and the slope of
this line gave a quantitative expression of the
isotope effect. The effect was the same whether the
amino acid was labeled in the 1 or 2 position, but
was entirely absent when the label was located
elsewhere. Further, it was less pronounced for
those amino acids with which adsorption is known

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

467

to play a relatively large part in exchange be-
havior. On the basis of these considerations, it was
possible to predict the magnitude of the isotope
effect for randomly labeled amino acids; excellent
agreement was obtained between the calculated
and observed values. The results indicate that the
effect of labeling on exchange behavior is unre-
lated to mass per se, but is related to the ionic
state of the molecule.

1520. Xenon and chloroform transfer between
blood and brain in the dog. C. B. PiTtTInGER,
H. L. Conn, Jr.* anp R. M. FEATHERSTONE.
Dept. of Pharmacology and Dept. of Surgery, Div.
of Anesthesiology, College of Medicine, State
Univ. of Iowa, Iowa City, and the Med. Dept.,
Brookhaven Natl. Lab., Upton, N.Y.
Radioactive xenon and chloroform were pre-

pared by neutron bombardment of these anes-

thetic agents within the nuclear reactor at

Brookhaven National Laboratory. Saturation and

desaturation processes within the dog brain were

studied with a technique employing a scintillation
counter for the intermittent, rapid determination
of radioactivity in blood from a carotid artery and
the sagittal sinus. Two rate constants, k; and ke,
were derived for the saturation and desaturation
processes with each of the agents. The k; values
for xenon and chloroform in both processes were

of a similar order of magnitude and averaged 1.15.

The ke values for the 2 agents agreed in both proc-

esses and averaged 0.20. These pairs of constants

suggest that the brain in saturation and desatura-
tion processes functions as a 2-compartment sys-
tem, presumably gray and white matter. The
similarity of the pairs of constants for xenon and
chloroform indicate that transference of these
substances from blood to brain is essentially a
flow-limited process not significantly influenced by
factors such as differences in diffusion or perme-
ability rates, or in modes of bonding. Differences
in postanesthetic recovery rates from the 2 agents
is not due to differences in their rates of transfer-
ence between blood and brain, but rather to differ-

: —A
ences in their excretion rations, ———— , at the

blood-air interface in the lungs as demonstrated
by their desaturation curves.

1521. Blood levels of hydantoins and pheno-
barbital in epileptics. G. L. Puiaa,* C. H.
Hine anv Tuos. L. Netson.* Dept. of Pharma-
cology and Exptl. Therapeutics, Univ. of Cali-
fornia, San Francisco, and Sonoma State Hosp.,
State Dept. of Mental Hygiene.

Blood levels of hydantoins and phenobarbital
were determined in patients receiving therapy for

control of epilepsy for periods ranging from 1

468

month to 4 yr. The method used was that reported
previously by this laboratory (Federation Proc. 14:
1225, 1955). The number of patients, dosage of
therapeutic agent, and blood level are summarized
in tabular form:

Daily Dose
Therapeutic (mg/kg) Blood Level (mg %) No. of
Agent Mean Range Mean Patients
Diphenylhydantoin 4.2 0.21.4 0.8 2
Mesantoin 2.8 0.6 1
Phenobarbital 13 1.43.2 2.2 3
Diphenylhydantoin 3.9 0.0-1.6 0.7 I
plus phenobar- 41° 0060 34"
bital
Mesantoin plus 4.2 0.6-1.6 0.9 ,
phenobarbital 3.6 0.7-5.2 2.3 e
Diphenylhydantoin 3.2 0.31.0 0.7 2
plus mesantoin 2.3
Diphenylhydant oin 3.3 081.5 1.2 9
plus mesantoin 3.3 5.27.7 65
plus phenobar-_ 6.1 dink i
bital

In general, the blood level of the drug was propor-
tional to the dosage. Hydantoin levels did not ex-
ceed 1.6 mg % and were consistently lower than
phenobarbital levels. Three patients who showed
evidence of phenobarbital intoxication (ataxia
and slowness) had levels greater than 5.0 mg %. A
single patient showing hyperplasia of the gums
had a hydantoin level of 1.5 mg %. (Supported, in
part, by the Breon Fund, granted through the
Committee on Med. Research, Univ. of California
Med. Ctr., San Francisco.)

1522. Influence of methyl-a-phenyl-2-piperi-
dine acetate hydrochloride on the pharma-
cological actions of reserpine. A. J. PLuM-
MER, R. A. Maxwe.u,* A. E. Earu* ann R.
RutLepGe.* Research Dept., Ciba Pharmaceuti-
cal Products Inc., Summit, N. J.
Methyl-a-phenyl-2-piperidine acetate hydro-

chloride (Ritalin), a central nervous stimulant

described by Meier, Gross and Tripod (Klin.

Wschr. 32: 445, 1955) has a modifying action on

various actions of reserpine. Normotensive dogs

are brought under the tranquilizing influence of

reserpine .by the daily oral administration of 50-

100 wg/kg of the drug. Such unanesthetized ani-

mals are in a state of quietude and exhibit miosis,

relaxation of the nictitating membranes, hypoten-
sion and bradycardia during the entire laboratory
day. The oral administration of 5 mg/kg of Ritalin

renders these animals active and alert within a

period of 30 min. and produces an almost complete

reversion of the nictitating membranes and pupils
to their normal state. After 3 hr. the effect of Ri-
talin disappears and the animals assume the ap-
pearance typical of reserpine action. Ritalin does
not antagonize the hypotensive effect of reserpine
measurably, however. Since Ritalin does not in-
fluence the nictitating membrane relaxation fol-

FEDERATION PROCEEDINGS

Volume 1§

lowing ganglionic blockade, it appears that it must
be acting at or near the site of action of reserpine
within the central nervous system by a rather spe-
cific type of antagonism. It is possible to inhibit
certain of the centrally mediated effects of reser-
pine by means of Ritalin without influencing the
hypotensive mechanism.

1523. Pentobarbital distribution relation.
ships in blood and tissues of dogs. GEORGE
A. Porter,* Joun C. Misko* anp Norman A,
Davin. Univ. of Oregon Med. School and Multno-
mah County Coroner’s Office, Portland.

Current methods of toxicologic analysis utilize
blood instead of tissues for extraction and spectro-
photometric determination of barbiturates
(BrackETT, JR. AND BrapFrorp, San Jose, Calif,,
Annals of Forensic Med., 1954. To be published),
While values are established for dose-drug rela-
tionships of barbiturates in tissues no data, except
for phenobarbital, are available for comparative
tissue-blood levels nor for distribution in whole
blood, red cells and plasma. When it is necessary to
identify barbiturates in embalmed or decomposed
tissues blood clots may be used but the possibility
of using bone marrow in such cases requires inves-
tigation. We have undertaken studies to provide
more data along this line. In 13 dogs given an aver-
age of 50 mg/kg of sodium pentobarbital i.p.,
analysis of 21 blood samples taken at hourly in-
tervals resulted in an equal distribution of drug
between plasma and red cells. From 85% to 90%
of the pentobarbital found in whole blood was re-
covered in the plasma and cells. Using the Dixon
sign test for paired differences the net difference
between calculated recovery (based on whole
blood) and theoretical recovery (plasma, red cell
analysis and hematocrit) was 22.18 with a prob-
ability greater than .25. Analysis of serial samples
showed a gradually declining barbiturate level. In
4 autopsied dogs the barbiturate C.S.F. values

were lower than those for blood while those forf i

bone marrow (femur) were quite low but still
within the quantitative range of the method.

1524. Chlorpromazine: a possible mechanism
of action. James B. Preston (introduced by
G. K. Mog). Dept. of Physiology, State Univ. of
New York, Syracuse.

The effect of chlorpromazine in the cat was stud-
ied on the monosynaptic and polysynaptic reflex
ares of the spinal cord, the ascending somato-
sensory system, the cerebral cortical arousal pat-
tern following stimulation of the ascending reticu-
lar activating system, and the spontaneous and
evoked discharges on the isolated cerebral cortex,
Doses of chlorpromazine exceeding those which
caused obvious changes in behavior of intact um
anesthetized cats produced no observable changes
in any of these preparations, with the possible ex-

plume 1§

t it must
‘eserping
ther spe-
0 inhibit
of reser-
icing the

elation.

GEORGE
RMAN A,
| Multno-

is utilize
spectro-
viturates
e, Calif,
blished),
rug rela-
a, except
parative
in whole
essary to
omposed
»ssibility
es inves-
) provide
an aver-
ital i.p.,
ourly in-
. of drug
o to 90%
d was re-
he Dixon
jifference
n whole
_ red cell
| a prob-
| samples
level. In
’, values
those for
but. still
10d.

thanism
luced by
Univ. of

vas stud-
tic reflex

somato-
usal pat-
ig reticu-
cous and
il cortex.
se which
itact unr
» changes
ssible ex-

March 1956

ception of. the cortical arousal pattern following
stimulation of the medial reticular formation.
Therefore, the scope of study was widened by a
simultaneous sampling of a number of cortical and
subcortical areas by means of gangs of electrodes
oriented stereotaxically. Chlorpromazine in doses
exceeding the minimal effective dose for behavior
changes in the intact unanesthetized animal
produced isolated seizure activity in the amyg-
daloid nuclear complex. With larger doses of
chlorpromazine activity changes of lesser magni-
tude were noted in other areas of the brain stem
and with still larger doses synchronized spike
activity, typical of ‘grand-mal’ type seizure
discharges, appeared throughout the brain stem
and cerebral cortex. The relative sensitivity of
the amygdaloid spontaneous activity to chlor-
promazine as compared to other cortical and brain
stem areas suggests a possible mechanism of
action, for the baso-lateral amygdaloid complex
has been suggested by others to mediate an
inhibitory influence over wide areas of the brain
stem.

1525. Chemical assay of epinephrine and
norepinephrine in plasma. Henry L. PRICE
AND ARTHUR DET. VALK, JR.* Dept. of Anes-
thesiology, Hosp. of the Univ. of Pennsylvania,
Philadelphia.

The use of chemical methods for the detection
of epinephrine (E) and norepinephrine (N) in
plasma has become popular, but their reliability
has not been established. The specificity of 2
chemical methods for the detection of E and N
has accordingly been studied. The 1st method
employs condensation of E and N with ethylene-
diamine, fluorimetric assay and separation of E
and N by a calculation depending upon measure-
ment of the emission spectra of the condensates
at two wave lengths (method of Weil-Malherbe
and Bone). The 2nd method—also fluorimetric—
is a modification of Lund’s technique for the assay
of adrenochrome and noradrenochrome. Both
methods employ adsorption of the plasma amines
on alumina, with subsequent elution in acetic
acid. In addition to E and N, dihydroxyphenyl-
alanine (DOPA), 3-hydroxytyramine (dopamine),
3,4 dihydroxyphenylacetic acid (‘dopac’), and
other dihydroxyphenyl compounds are adsorbed
from plasma on alumina and are eluted in acetic
acid. These substances condense with ethylene-
diamine under the conditions described by Weil-
Malherbe and Bone, and form fluorescent com-
pounds which may interfere with the assay of E
and N. The Lund method detects only catechol
amines with a hydroxyl group at position 2 of the
carbon side chain, so that adrenaline and nor-
adrenaline—but not dopa, dopamine, or ‘dopac’—
are assayed by this method. Comparison of plasma
eluates by the 2 methods reveals that most of the

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

469

fluorescence detected by the Weil-Malherbe and
Bone method does not result from the presence
either of E or of N, but may result from the pres-
ence of ‘dopac’, a supposed metabolite of dop-
amine. A modification of this method may be
satisfactory for the determination of E but
not of N.

1526. Effects of several types of antihyper-
tensive agents on the v tor resp to
tilt in dogs (orthostatic hypotension).
Nicuouas A. Prioui* anp Martin M. WINBURY.
Research Dept., Schering Corp., Bloomfield, N. J.
The purpose of this study was to compare the

effects of several antihypertensive agents on the

vasomotor response to tilt at doses sufficient to
produce hypotension. The agents studied are
classified according to the mechanism of action,
as follows: ganglion blocking agents—tetraethyl-
ammonium, hexamethonium and trimethaphan

(Arfonad); adrenergic blocking agents—azapetine

(Ilidar) and benzazoline (Priscoline); vasodila-

tors: pentaerythrityl tetranitrate and mannitol

hexanitrate and compounds with mixed action:
hydrazinophthalazine (Apresoline). The typical
response to control tilts of morphine-Nembu-
talized dogs was an initial fall in aortic pressure
followed by partial recovery while still in the
vertical position (30-60 sec.) and ‘overshoot’ on
return to horizontal. Complete blockade of the
vasomotor response to tilt was typified by absence
of recovery of aortic pressure while vertical and
absence of ‘overshoot’ on return to horizontal.

Complete blockade was observed with the gan-

glion blocking agents and the adrenergic blocking

agents. After Apresoline, partial blockade of the
response to tilt was observed i.e. the aortic pres-
sure recovered while the animal was vertical but

‘overshoot’ on return to horizontal was absent.

The vasodilators did not appear to block the

vasomotor reflexes.

1527. Action of ryanodine on the cardio-
vascular system of the cat. LEONARD PRo-
cita AND Matruew Kutvz (introduced by
Erwin E. Ne son). Dept. of Pharmacology, St.
Louis Univ. School of Medicine, St. Louis, Mo.
Previous studies (Procita, SHIDEMAN AND

Ratusun. J. Pharmacol. & Exper. Therap. 1061:

411, 1952) have shown that ryanodine is highly

toxic to the heart of the anesthetized and arti-

ficially respired dog. In the present study ryan-
odine was injected into anesthetized (pentobarbi-
tal sodium 30 mg/kg) artificially respired cats
through polyethylene catheters into the jugular
and femoral veins, the central and peripheral ends
of the carotid arteries, central and peripheral
ends of the femoral arteries and the vertebral
artery. Regardless of the route of administration
200-500 ng/kg of ryanodine produced a pronounced

470

fall of blood pressure. The time of onset of the
hypotension depended on the vein or artery into
which the drug was injected. It occurred, for
example, 10 sec. after injection into the femoral
vein and from 45 to 60 sec. following injection
into the vertebral artery. The hypotension is not
influenced by vagotomy or atropine. It also occurs
in animals with spinal cord transections at C,-C2
although the amount of drug:. quired is of a lesser
order. Electrocardiographic evidence indicates
that the fall of blood pressure is independent of
alteration in the heart rate. Ryanodine does not
alter the pressor response to either carotid clamp-
ing or asphyxia or to injected epinephrine. In
open chest cats an initial injection of ryanodine
of over 600 ug/kg produces cardiac failure which
is neither prevented nor reversed by ouabain
although the heart is still responsive to epi-
nephrine. If the animal recovers from an initial
hypotensive dose of ryanodine, further injections
of the drug, in doses of the same magnitude or
greater, do not produce a similar hypotensive
effect. This rapid development of refractoriness
to the action of ryanodine is long persisting.
(Supported by a grant from the St. Louis Heart
Assoc.)

1528. Oxygen effects on digitoxin. C. D.
Proctor, JoHN REBAR, JR. AND BLANCHE
TIGERMAN ReEBAR (introduced by Y. T.
OrsTER). Dept. of Pharmacology and Exptl. Thera-
peutics, Stritch School of Medicine and the Grad.
School, Loyola Univ., Chicago, Ill.
Polarographic studies have revealed that digi-

toxin is capable of taking up dissolved oxygen

from solution. A peroxide product is formed and
the rate of reaction is accelerated by hemolyzed
blood. Formation of this product is more favored
by K* and Mg** than by Nat, while Ca**, Co*+
and Mn* were without effect. Evidence has also
been obtained demonstrating digitoxin complex-
ing effect on molecular oxygen. These findings
have been correlated with in vitro inhibitory
effects of digitoxin on heart adenosinetriphospha-
tase and cholinesterase. At stoichiometric equiva-
lent concentration to digitoxin the peroxide
product effects a higher degree of inhibition of
these enzymes than that produced by digitoxin.

1529. Neuromuscular and ganglionic blocking
actions of NHI-196, a hypotensive alkaloid
derived from Ormosia panamensis. GERTRUDE
P. Quinn, Witui1AM M. But er, JR. aNnp NEIL
C. Moran (introduced by BERNARD B. Broptie).
Lab. of Chemical Pharmacology, Natl. Heart
Inst., Bethesda, Md.

NHI-196, a quaternary-like base derived from
the seeds of Ormosia panamensis produces neuro-
muscular and ganglionic blockade. With peroneal
nerve-anterior tibial muscle preparations in anes-

FEDERATION PROCEEDINGS

Volume 1§

thetized dogs, the maximal twitch responses to
periodic stimulation of the nerve were recorded,
Single intra-arterial injections of 0.7-4.0 mg of
NHI-196 produced progressive increases in the
degree of blockade. On i.v. administration, 10
times the hypotensive dose was required to
produce blockade. Neostigmine antagonized the
blockade. In these same preparations 0.15 mg of
d-tubocurarine chloride i.a. produced 100%
blockade. NHI-196 produced a partial depression
of the response to direct muscle stimulation but
no greater than that produced by succiny] choline
or d-tubocurarine chloride. Confirmation of a
competitive-type blockade was obtained with
chickens in which NHI-196, 2 mg/kg, produced
a flaccid paralysis in contrast to the contracture
produced by decamethonium, a depolarizing
agent. The rabbit ‘head-drop’ was obtained by
the i.v. administration of 1.1-1.9 mg/kg of NHI-
196 compared to 0.14 to 0.2 mg/kg of d-tubocura-
rine chloride. The bradycardia produced by
peripheral vagal stimulation or by the injection
of acetylcholine was diminished by 1.5 mg/kg of
NHI-196 in dogs. Complete blockade of peripheral
vagal stimulation and of the vasopressor response
to tetramethylammonium was produced by NHI-
196 with doses approximately 100 times the
hypotensive dose. The effects of acetylcholine
were not completely blocked. In concentrations
exceeding those necessary to cause a vasodepres-
sion, NHI-196 is a competitive type neuromus-
cular blocking and a ganglionic blocking agent.
(NHI-196 provided by Drs. Helen A. Lloyd and
Evan C. Horning of the Lab. of Chemistry of
Natural Products, NHI.)

1530. Influence of incubation of polysac-
charide with serum on pyrogenicity. Davip
P. Ratu, J. R. Gaskins anp MAarGaret G,
KELLY (introduced by CHaRLEs G. ZuBrop).
Natl. Cancer Inst., Bethesda, Md.

Previous reports indicate that incubation of
plasma or serum with pyrogenic bacterial poly-
saccharide prior to injection can yield an aug-
mented febrile response, particularly in tolerant
rabbits. This report concerns the effect of varying
the amount of serum and the time of its incubation
with polysaccharide on pyrogenic activity. Serum
was obtained aseptically from rabbit heart blood
and was used within 2 hr. The pyrogen was lot
P-35 of a polysaccharide from S. marcescens pre-
pared by Perrault and Shear. The response meas-
ured was maximum post-pyrogen increment in
rectal temperature. Either 10 parts pyrogen in
saline to 1 part serum, or equal parts of each were
incubated at 37°C for 10, or 60 min. and given i.v.
to fresh rabbits. Suitable controls were also used.
In rabbits made tolerant by 7-13 daily injections
of pyrogen crossover experiments were performed.
Each animal received either equal parts of pyrogen

ume 1§

nses to
corded,
mg of
in the
ion, 10
red to
red the
) mg of

10%
ression
ion but
choline
n of a
1 with
oduced
racture
arizing
ned by
f NHI-
ocura-
ed by
jection
t/kg of
ipheral
sponse
, NHI-
es the
choline
rations
lepres-
romus-
agent,
yd and
stry of

lysac-
Davi
ET G,
BROD),

ion of
| poly-
n aug-
olerant
arying
bation
Serum
; blood
vas lot
ns pre-
» meas-
ent in
gen in
h were
‘en i.v,
) used.
>ctions
ormed.
yrogen

—-

March 1956

in saline and serum, or pyrogen in serum alone
incubated either 20, or 60 min., as well as pyrogen
in saline similarly incubated. Fresh rabbits given
equal parts of pyrogen in saline and serum incu-
bated for 60 min. responded with less fever than
the controls; all other groups, however, were in-
distinguishable from the controls. Tolerant rab-
bits, when given any combination of serum
incubated pyrogen responded with less fever
than when given pyrogen in saline. These data are
considered evidence that, under certain condi-
tions, prior incubation of polysaccharide with
serum can reduce pyrogenicity.

1531. Metabolism of C'™-formate by spleen
breis from normal and x-irradiated rats.
D. A. Rapporort,* R. A. SEIBERT AND V. P.
Couuns.* Depts. of Biochemistry, Pharmacology
and Radiology, Baylor Univ., College of Medicine,
Houston, Tex.

A study was initiated to determine the effects
of lethal, total body x-radiation on the metab-
olism of C'4-formate by isolated rat tissues. Rats
were exposed to total body radiation of 2000r
and 5000r, respectively, and 15 hr. later the spleens
were removed. Simultaneously, spleens were also
removed from unirradiated rats as controls.
Minced spleens were then incubated with 5 um
C'-formate, 10 um each of glycine, glutamine
and glucose in Krebs-Ringer phosphate buffer
for 1 hr. at 37°C in oxygen. The products from the
incubations were separated and the distribution
of C4 determined. The results indicated that total
body irradiation of rats significantly reduced the
incorporation of C'-formate into acid soluble
components (tentatively identified as serine and
cysteine), and into lipids and nucleoproteins.
Oxidation of C'4-formate to carbon dioxide was
also reduced in spleens from these animals. Similar
experiments with minced preparations of liver,
kidney, testes, and intestine from irradiated rats
showed that they were much less sensitive to
radiation than the spleen. (These investigations
were supported in part by the American Cancer
Society Grant R19B through the recommendation
of the Natl. Research Council.)

1532. Survival time of rats with damaged livers
after severe hemorrhage. G. Cari Rav anp
EMANUEL A. RosEN (introduced by Lesure L.
EIsENBRANDT). Dept. of Pharmacology, Univ. of
Kansas City, Kansas City, Mo.

Rau (Am. J. Physiol. 156: 454, 1949) demon-
strated a very early rise in blood hypertensinogen
in hemorrhaged dogs at from 16 to 32 min. Since
hypertensinogen is formed in the liver it seemed
necessary to determine the importance of the
Renin-Hypertensin mechanism to the animal
during time of stress. To reduce blood hyper-
tensinogen the liver was damaged by an oral dose

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

471

of carbon tetrachloride (3 ml/kg of a 40% solution
in olive oil), Twenty-four hr. later the rats were
bled to 40% hemorrhage by cannulation of the
carotid artery. It has been shown in this labora-
tory that with the above dosage maximal liver
necrosis takes place at 24 hr. Blood pressure and
survival time were compared with normal rats
bled by the same procedure. Thirty Sprague-
Dawley male rats weighing from 298 to 320 gm
were used. During tests they were kept in a con-
stant temperature box at 39°C. Before hemorrhage
blood pressures in both control and experimental
animals determined by the microphonic manom-
eter varied from 130 to 140 mm Hg, when all rats
were anesthetized with sodium pentobarbital.
With liver damaged animals the dose was de-
creased to a minimal. The anesthetic was not a
factor in determining survival. Control animals
survived an average of 109 min. while the 24-hr.
carbon tetrachloride treated rats survived an
average of 31 min. Control rats survived 3.60 times
longer than treated rats. Determinations of blood
hypertensinogen in the 2 groups of animals are
in progress.

1533. Digitoxin effects on cholinesterase from
dilated heart muscle. BLANCHE TIGERMAN
Respar, JoHN ReBar, JR. AND C. D. Proctor
(introduced by Y. T. OgsteR). Dept. of Pharma-
cology and Exptl. Therapeutics, Stritch School of
Medicine, and Grad. School, Loyola Univ., Chicago,
Til.

Work carried out by us prior to this communica-
tion has indicated an in vitro inhibitory effect of
digitoxin on heart cholinesterase (CHase). The
kinetics of this inhibition are such that the com-
plex formed between digitoxin and the CHase is
either irreversible or pseudo-irreversible. As a
part of studies designed to yield information on
possible digitoxin action at the molecular-cellular
level, the in vitro effects of this glycoside on
CHase derived from dilated heart muscle has
been compared with the effect of the compound
on undilated heart muscle. Experimental cor
pulmonale, produced in the pentobarbitalized
dog by constriction of the pulmonary artery with
a modified Potts clamp, was effected so that
observable dilation of the right ventricle with
rise in venous pressure was produced without
observable dilation of the left ventricle. Enzyme
homogenates made from the muscle of the respec-
tive ventricles were used as CHase sources and
acetylcholine as substrate in experiments designed
to demonstrate digitoxin in vitro effect on the
enzyme. Similar studies were made on the ven-
tricles from normal pentobarbitalized dogs.
Findings indicate that there is no significant
difference in the degree of inhibition produced by
digitoxin on CHase derived from dilated heart

472

muscle and on that derived from undilated heart
muscle.

1534. Digitoxin effects on ATPase from dilated
heart muscle. JoHN REBAR, JR., BLANCHE
Resar anp C. D. Proctor (introduced by
Y. T. Oxstrer). Dept. of Pharmacology and
Exptl. Therapeutics, Stritch School of Medicine,
and Grad. School, Loyola Univ., Chicago, Ill.
Prior work carried out by us has indicated an

in vitro inhibitory effect of digitoxin on heart
adenosinetriphosphatase (ATPase) under speci-
fied conditions of substrate concentration in the
enzyme reaction mixtures. The kinetics of this
inhibition are such that the complex between
digitoxin and the ATPase is reversible in nature
and the degree of digitoxin inhibition is regulated
by the concentration of the substrate, adenosine-
triphosphate (ATP). In order to study the effect
of decreased endogenous muscle ATP on this
in vitro phenomenon, the effect of digitoxin on
ATPase derived from dilated heart muscle and
from undilated heart muscle has been compared.
Experimental cor pulmonale was produced in the
pentobarbitalized dog by constriction of the pul-
monary artery with a modified Potts clamp. The
preparation was controlled so as to produce
dilation in the right ventricle with a rise in venous
pressure but no observable dilation in the left
ventricle. Enzyme homogenates from the muscle of
the respective ventricles furnished ATPase sources
for the study of digitoxin effect on ATPase ac-
tivity. Similar studies were made on ventricles
from normal pentobarbitalized dogs. In all cases
the ATP level of each ventricle was determined,
correcting for inosinetriphosphate. Results in-
dicate that the degree of digitoxin ATPase inhibi-
tion is greater for ATPase derived from dilated
heart muscle than it is for enzyme obtained from
undilated heart muscle. Findings indicate further
that this effect is probably correlated with com-
paratively lower endogenous ATP levels found in
the dilated heart muscle.

1535. Neuromuscular blocking action of
Prestonal in anesthetized man. L. RENDELL-
BakeER,* Joun H. Brrcew,* PETER B. D’Sovuza*
AND Francis F. Foupss. Dept. of Anesthesiology,
Mercy Hosp., Section on Anesthesiology, Dept.
of Surgery, Univ. of Pittsburgh School of Medi-
cine, Pittsburgh, Pa.

The effects of Prestonal (N,N, N’-tetramethyl-
N-N’-bis-carbopropoxymethyl-3,14-dioxa - hexa-
decane-1,16-diammoniumbromide) was - investi-
gated on the tidal exchange of 31 patients operated
under low spinal anesthesia. For the patient’s
comfort light general anesthesia was maintained
during the test. After tidal volume (measured
with a Bennett ventilation meter) and respiratory
rate became stabilized 1.5 mg/kg Prestonal was

FEDERATION PROCEEDINGS

Volume 1§

injected intravenously in 30 sec. Apnea, lasting
308+10 sec. developed in 88+.5 sec. Tidal volume
returned to control value in 730+34 sec. 0.75 mg/
kg Prestonal given at this time produced apnea
lasting 27117 sec. in 65+2 sec. Tidal volume
became normal again in 540+30 sec. A second
0.75 mg/kg dose of Prestonal had at this time
almost identical effects on onset and duration of
apnea. Thei.v. injection of 0.3 mg/kg edrophonium
at the time when tidal volume became 50% of the
control value, however, caused apnea in 7 out of
9 patients in 30-60 sec. and delayed the return of
tidal volume to control value for 730+-50 sec,
Pyridostigmine in 0.15 mg/kg doses administered
the same way caused no reduction of tidal volume
but delayed the return to control value. In 7 pa-
tients the same dose of edrophonium given when
tidal volume reached control value caused apnea
in 2, and 70-95% reduction of tidal volume in §,
Return to control value took an average of 240 sec.
The potentiating effect of edrophonium and pyri-
dostigmine on Prestonal suggests that it produces
a depolarization block in man.

1536. Secretion of choline by renal tubules in
the chicken. BARBARA R. Rennicx. Physiology
Dept., State Univ. of New York, Med. College,
Syracuse.

The active renal tubular transport of the bases
n-methylnicotinamide and tetraethylammonium
has been demonstrated in the dog and chicken.
This communication reports the active transport
of choline by the renal tubules of the chicken.
Choline chloride 1 mg/min. and paraminohippu-
rate (PAH) 90 yug/min. were infused simul-
taneously into a vein supplying the renal-portal
circulation which bathes unilaterally the renal
tubular cells. Urine collected individually from
each ureter showed a recovery of 30% of the in-
fused amount of choline in the urine from the
kidney of the injected side. Urine from the control
side contained no detectable choline. Choline was
determined by the Reineckate method. About
70-90% of the PAH appeared in the urine from
the infused kidney, indicating an effective renal-
portal circulation. When the basic cyanine dye
(1’-ethy]-3 ,6-dimethy]-2-phenyl-4-pyrimido-2’ cy-
anine chloride) was added to the above infusion
the secretion of choline from the infused side
ceased.

1537. Necrotizing effect of pathologic human
sera on guinea pig skin. Evetyn F. Rep-
PLINGER, I. R. Scuwartz, H. S. BowMANn AND
L. M. Tocantins (introduced by C. P. Kraatz),
Charlotte Drake Cardeza Fndn., Jefferson Med.
College, Philadelphia, Pa.

Skin ulcerations are often observed in patients
with malignant lymphoma. Sera from such pa-
tients (with or without skin lesions) injected

ume 16

lasting
volume
75 mg/
. apnea
volume
second
is time
tion of
nonium
) of the
out of
turn of
50 sec,
istered
volume
n 7 pa-
n when
| apnea
1e in 5,
240 sec.
d pyri-
‘oduces

ules in
jstology
College,

e bases
10onium
hicken.
unsport
hicken.
yhippu-
simul-
-portal
2 renal
y from
the in-
om the
control
ine was
About
e from
. renal-
ne dye
9-2’ ey-
nfusion
sd side

1uman
', REp-
AN AND
RAATZ).
n Med.

atients
ich pa-
njected

March 1956

intracutaneously into guinea pigs produce necro-
sis and uléeration. The necrotizing factor is
nondialyzable, destroyed at 50°C, lost on 2-fold
dilution, active only at pH 5.5-8.5 and well pre-
served by freeze-drying. The factor has not been
found in the serum of normal healthy subjects,
and seems analogous to that described in the sera
of rheumatoid arthritis patients by Boake and
Lovell (Brit. J. Exper. Path. 35: 345, 350, 1954).
Because of the lability of the factor, electro-
phoretic separation has been unsuccessful. At-
tempts to relate this factor to Kidd’s (J. Exper.
Med. 98: 565, 1953) guinea pig serum anti-mouse-
lymphoma factor, seem to indicate that normal
human serum can neutralize the anti-tumor effect
of guinea pig serum to some extent, and that
necrotizing serum is somewhat more active in this
respect than nermal human serum. The factor was
consistently found in high potency in the sera of
patients with Hodgkin’s disease, reticulum cell
sarcoma and mycosis fungoides. The sera of pa-
tients with lymphocytic lymphomas, chronic
monocytic and lymphocytic leukemias only occa-
sionally give positive results, while those of pa-
tients with chronic myelocytic and acute leu-
kemias have been almost always negative. This
necrotizing ability of the serum of patients with
lymphomas may be a manifestation of the resist-
ance of the host to the tumor.

1538. Effectiveness of Pagitane (cycrimine
hydrochloride) and Kemadrin (procyclidine
hydrochloride) in prevention of airsickness.
A, A. Renzi* anp Lawrence J. Mitcu. Dept.
of Pharmacology and Biochemistry, School of
Aviation Medicine, USAF, Randolph AFB,
Randolph Field, Tex.

Earlier studies (Mil. Surgeon 108:20, 1951) have
shown that Artane, an antispasmodic, was rela-
tively effective in preventing motion sickness.
In the present study we have determined the
effects of 2 newer antispasmodics, Pagitane and
Kemadrin, in preventing airsickness in individuals
subjected to simulated turbulence. For each
flight 32 volunteer airmen were divided into
4 groups. All medications were administered 1 hr.
prior to the actual start of the flight. Group 1
received capsules containing lactose, which served
as the placebo control. Group 2 received capsules
containing 5 mg of Pagitane while growp 3 were
given capsules containing 5 mg of Kemadrin.
Group 4, on the other hand, to insure a more accu-
tate comparison, received capsules containing
50 mg of the antihistamine Benadryl, a known
preventative of motion sickness. All groups were
distributed evenly throughout the aircraft. The
flight itself was of 60 min. duration and consisted
of set motion patterns which would effectively
produce emesis. The preparations were evaluated
on the basis of the subject’s emetic response.

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

473

Results based on 63 individuals per group indi-
cated that the incidence of sickness was highest
in the placebo group (38.1%) and that the inci-
dence in the Kemadrin-treated subjects (17.5%)
was far less than that in the Pagitane group
(28.5%). In terms of protection, Kemadrin was
54% effective while Pagitane was only 25% effec-
tive. Kemadrin compared very favorably with
Benadryl which itself showed 50% protection
against airsickness. The preparation demonstrated
no untoward side effects at the dose level
employed.

1539. Effect of sodium azide on action of
pentobarbital in mice. R. K. Ricwarps,
J.D. Taytor, JEANNE C. Davin* AND ELEANOR
M. Swenson.* Dept. of Pharmacology, Abbott
Labs., North Chicago, Ill.

It has been demonstrated that many barbitu-
rates are destroyed in the body, particularly by
enzymes contained in the microsomes of liver
cells. However, little is known about the nature of
the enzymes involved and the sequence of their
action. The azide ion is known to be a potent in-
hibitor of a number of enzymatic processes. The
action of Na azide upon the duration of action and
rate of metabolic degradation of pentobarbital
was: studied in several hundred albino mice. Na
azide given i.p. proved to have a very steep dose
mortality curve (LDis = 30 mg/kg; LD50 = 32 mg/
kg; LDs = 35 mg/kg). In doses of 5-15 mg/kg
no symptoms of toxicity were noticed, nor was
the LDs5o of i.p. pentobarbital influenced by prior
administration of Na azide, but the EDs of the
barbiturate was significantly lowered. A prolong-
ing effect on pentobarbital sleep was noticed with
doses as low as 5 mg/kg of Na azide; with 10 mg/kg
the sleeping time was increased about 100%. In-
jection of Na azide into mice awaking from pento-
barbital sleep resulted in a restoration of sleep
in a significant number of animals. The percentage
of mice responding in this manner was much
greater after small than after large doses of pento-
barbital, i.e., inversely proportional to the dura-
tion of the pentobarbital effect. No significant
difference was found at awaking time in the total
barbiturate content recovered from the bodies of
mice given pentobarbital with or without Na
azide. However, the azide treated animals slept
about twice as long. A comparison of the slopes of
the degradation curves of the barbiturate indi-
cates that the azide markedly retarded the meta-
bolic degradation of pentobarbital.

1540. Effect of carbon dioxide inhalation on
plasma concentrations of epinephrine and
arterenol. J. A. RICHARDSON AND E. F. Woops.*
Dept. of Pharmacology, Med. College of South
Carolina, Charleston, 8S. C.

The effect of carbon dioxide inhalation on

474

arterial plasma concentrations of epinephrine
and arterenol was determined by a fluorimetric
method (RicHaRpsoN, RICHARDSON AND BRODIE,
J. Lab. & Clin. Med. In press), in open-chest,
bilaterally vagotomized dogs under barbiturate
anesthesia. Heart contractile force and arterial
blood pressure were recorded in each experiment.
A carbon. dioxide-oxygen mixture of 25:75% was
administered by a nonrebreathing technic from
a Foregger anesthesia machine and a reservoir
bag. The average interval of exposure to the
carbon dioxide-oxygen mixture was about 10 min.
Blood samples were taken from the left femoral
artery. Consistent and substantial increments in
the concentration of plasma amines were noted
1) during the period of myocardial depression
produced by the gas, and 2) during the interval
of increased cardiac contractility following the
withdrawal of the carbon dioxide. These results
are compatible with the concept which recognizes
the cardiac depressant action of carbon dioxide
(BonIFACE AND Brown, Am. J. Physiol. 172: 752,
1953) and the opposing stimulant action of epi-
nephrine and arterenol. (Supported by research
grant H-1846 from the Natl. Heart Inst., Natl.
Insts. of Health, PHS.)

1541. Inhibition of accumulation of chroni-
cally ingested lead in rats by simultaneous
feeding of edathamil calcium disodium
(Na2CaEDTA). FREpRic RIEDERS AND JOAN
E. Corpetann.* Dept. of Pharmacology, Jefferson
Med. College, Philadelphia, Pa.

Two groups of weanling Sherman albino rats
were used. Group 1 received a diet containing
2 ppm lead, incorporated into food and drinking
water as lead acetate. The diet of group 2 con-
tained 760 ppm NasCaEDTA and 3 ppm lead,
this additional ppm of lead originating as an
impurity from the NasCaEDTA. The animals
were maintained on the respective diets for 38 wk.
with no gross or hematological ill effects. Then,
10 males and 10 females from each of the 2 groups
were killed by chloroforming. Stomach, intestine,
skin and tail were removed and discarded. The
entire refnainder of each animal was digested
and analyzed for its lead content by a dithizone
method. The lead contents of the animals were
as follows: (values are given in mg/100 gm; means
and standard deviations are reported) group 1,
males—0.049+0.025; females—0.054+0.028; growp
2, males—0.020+0.008; females—0.028+0.011. Ac-
cumulation of lead in the course of its ingestion
by rats is significantly diminished by the simul-
taneous ingestion of NasCaEDTA.

1542. Enhancement of urinary Cu excretion
in a case of hepato-lenticular degeneration
by intramuscular edathamil calcium di-

sodium (NasCaEDTA). Frepric RIEpDERs.

FEDERATION PROCEEDINGS

Volume 1§

Dept. of Pharmacology, Jefferson Med. College,

Philadelphia, Pa.

A girl, age 18, weighing 98 lb., with hepato-
lenticular degeneration of at least 3 yr. standing,
excreted during 4 successive 24-hr. periods the
following respective amounts of Cu in the urine:
(these, as well as subsequent values are reported
as mg Cu/I]. of urine, corrected to sp.gr. 1.024)—
0.208, 0.346, 0.332, 0.197. The patient was given
10 ml of a 10% aqueous solution of NasCaEDTA
(Calcium Disodium Versenate, Riker); 5 ml were
injected into each buttock. Subsequent urine
specimens, collected during the indicated time
intervals after the injection, contained the follow-
ing concentrations of Cu: 0 to 1.25 hr.—3.503,
1.25-3.5 hr.—2.845, 3.5-5.75 hr.—2.830, 5.75
7.5 hr.—2.009, 7.5-24 hr.—0.892, 24-48 hr.—0.740,
48-72 hr.—0.572. Similar injections were made at
weekly intervals during the following 7 wk. Urine
was collected for 24 hr. before and after each
injection and the following concentrations of Cu
were found: (in each instance the 1st figure repre-
sents the urinary Cu concentration in the pre-
injection specimen, the 2nd figure that in the
24 hr. specimen collected after the injection). 1—
0.345, 1.460; 2—0.224, 0.895; 3—0.098, 1.352; 4—
0.421, 1.750; 5—0.264, 0.842; 6—0.339, 1.180; 7—
0.283, 0.685. The preceding observations indicate
that the extent as well as the time course of the
enhancement of urinary Cu excretion following
intramuscular administration of NasCaEDTA
is similar to that seen after intravenous adminis-
tration of the drug.

1543. Nonsaponifiable components of malig-
nant human liver in the Penn serofloccula-
tion reaction. RicHarp F. Ritey, YOsHITsvG!I
Hoxama,* Paut Krarz* anp Harry S. Penn.’
Dept. of Radiology, Univ. of California, Med.
Ctr., Los Angeles.

Penn has described the preparation of an agent
which gives a positive seroflocculation reaction in
a high percentage of sera from cancerous humans.
The ‘antigen’ was prepared from the nonsaponifi-
able fraction of liver from humans which had
succumbed to cancer (J. Natl. Cancer Inst. 12:
1389, 1952). The total nonsaponifiable fraction,
freed of digitonin precipitable and ketonic mate-
rials, and apparently containing the bulk of
serologically active compounds, has been sub-
mitted to chromatographic fractionation on
silicic acid. It was separated into 4 major groups
of components. The first 2, which eluted with
pentane and with 2.5% ether in pentane were
serologically inactive hydrocarbons and were aot
further examined. The 3rd fraction, eluted slowly
by 7.5% ether in pentane, was a heterogenous,
serologically active mixture of alcohols. Partition
into complexing and noncomplexing fractions
with urea provided 2 subfractions which were

a SE «ae lll oe i es ee a ee

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ob

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College,

hepato-
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ods the
2 urine:
eported
1.024)—
S given
aEDTA
ml were
t urine
d time
follow-
—3.503,
, 5.75
—0.740,
nade at
:. Urine
er each
s of Cu
2 repre-
he pre-
in the
ym). J—
52; 4-
80; 7—
ndicate
: of the
llowing
.EDTA
dminis-

malig-
recula-
{ITSUGI
Penn.*
1, Med.

n agent
‘tion in
umans.
uponifi-
ch had
ust. 12:
‘action,
; mate-
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n sub-
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March 1956

resolved into their components by reverse phase
partition ‘chromatography. Hexadecanol, oc-
tadecanol and traces of higher aliphatic alcohols
were identified. An unsaturated aliphatic alco-
hol(s), a secondary alcohol and at least one
branched and/or alicyclic alcohol were present.
The 4th group of components eluted from silicic
acid by 25 to 75% ether, was a complex mixture of
serologically active and inactive components.
The infra red spectra of a number of presently
unidentified components from this fraction have
been compared. The relative intensities of the
bands at 7.35 and at 6.8 u suggests that several
components of this fraction are branched or
methyl substituted alicyclic alcohols.

1544. Pharmacology of N!-(n-Butylearbamyl)
sulfanilamide. Mary A. Root anp R. C.
AnpERSON. Lilly Research Labs., Indianapolis,
Ind.

N!-(n-Butylcarbamy]) sulfanilamide (Substance
BZ-55) has been reported to cause hypoglycemia
when administered by mouth and to be effective
in the treatment of certain cases of diabetes
mellitus (Deutsche med. Wchnschr. 80: 1449, 1955).
In mice the intravenous LD50 is between 1.4 and
2.0 gm/kg. Doses of 2.5-3.0 gm/kg intraperi-
toneally in rats cause decreasing activity and
death in 3-10 hr. Oral administration of 1.0 gm/kg
in rats causes fatal hypoglycemia in some animals.
In rabbits doses of 1.0-1.5 gm/kg orally are lethal
unless the hypoglycemia is treated with glucose.
In dogs 0.7-1.0 gm/kg orally causes vomiting,
muscle twitching and weakness with little decrease
in blood glucose concentration. Hypoglycemia
occurs upon intravenous administration of this
substance to rabbits in doses as low as 50 mg/kg.
Oral administration of doses of 0.5-1.0 mg/kg in
rats and rabbits produces a significant fall in
blood glucose concentration and may cause hypo-
glycemic depression and convulsions. In normal
dogs even toxic doses may cause very little hypo-
glycemia. Daily treatment of alloxan diabetic
rats, rabbits and dogs with substance BZ-55 did
not alleviate the diabetes. In a diabetic dog the
combination of substance BZ-55 and insulin was
more effective than insulin alone in lowering blood
glucose concentration and in decreasing urinary
sugar excretion. Single doses of 2-4 gm orally in
a 10 kg pancreatectomized dog did not cause any
decrease of blood glucose.

1545. Pharmacological and toxicological ac-
tions of heliotrine. C. L. Rose,* P. N. Harris
AND K. K. Cuen. Lilly Research Labs., Indian-
apolis, Ind.

Heliotrine, an alkaloid belonging to the pyrrol-
izidine group, was isolated from the plant Helio-
tropium lasiocarpum by Menshikov (1932). It was
obtained later by Price and his colleagues of

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

475

Melbourne (1954) as one of the principal bases
from Heliotropium europeum L. Like lasiocarpine,
extracted from the same plant and on which we
reported earlier, heliotrine is a hepatotoxic alka-
loid. Intraperitoneal doses of 250 mg/kg produced
central necrosis of the liver in rats. The same dose
in mice caused necrosis of the liver, but this was
not so well defined and central necrosis was not
always present. The Lpso+standard error, by
intravenous injection in mice, was found to be
150.7+9.9 mg/kg. Five mice that survived in-
travenous doses of 160 mg/kg showed normal
tissues 10 days after injection. In 90-day old rats,
intraperitoneal injections of 100 mg/kg caused a
retardation of prothrombin time within 2 hr., a
maxima! effect in 12 hr., with a return to normal
in 30 hr. Concentrations of 1-20,000 or 1-10,000
had no action on the isolated uterus of the rabbit
or the guinea pig but relaxed the isolated rabbit
ileum without loss of amplitude of contraction.
A concentration of 1-10,000 relaxed the guinea
pig ileum and one of 1-100,000 partially released
the methacholine-induced spasm of the same
organ. In anesthetized dogs, a 5 mg/kg dose in-
jected intravenously caused no changes in blood
pressure, respiration or electrocardiogram. Two
minutes after injection in the same preparations
the intestinal movements were greatly augmented.

1546. Effect of sodium polyanhydroman-
nuronic acid sulfate on incidence of ulcers
in the Shay rat. Harry Rosen,* Partricta
TOWNSEND* AND JOSEPH SEIFTER. Wyeth Inst.
for Med. Research, Radnor, Pa.

It has been reported previously (LEVEY AND
SHEINFELD, Gastroenterology, 27: 625, 1954) that
heparin, chondroitin sulfate and Paritol-C (a
clinical grade of sodium polyanhydromannuronice
acid sulfate) inhibit the proteolytic action of
pepsin in vitro and that chondroitin sulfate
markedly reduced the incidence of ulcers in the
Shay rat. It seemed of interest to us to investigate
the effects of sodium polyanhydromannuronic acid
sulfate alone and in combination with alumina
gel, on ulcer formation in the Shay rat. The ani-
mals were fasted for 48 hr., the pylorus of each
rat ligated and drugs administered orally after
lavage of the stomach. Nineteen hours later the
animals were killed and the stomachs examined
for ulcers. Sodium polyanhydromannuronic acid
sulfate and alumina gel, both provide significant
protection from ulcers and mixtures of the 2 re-
duce ulcer formation to a greater degree than
either alone.

1547. Effect of nikethamide in combatting
anticholinesterase poisoning. PH1Lip Rosen-
BERG* AND J. M. Coon. Dept. of Pharmacology,
Jefferson Med. College, Philadelphia, Pa.
Nikethamide plus atropine sulfate protected

476

against ethyl-p-nitrophenylthionobenzenephos-
phonate (EPN) poisoning in female rats when
administered immediately following EPN, but
nikethamide alone did not, confirming the findings
of DiStefano et al. 1951. Further studies have
shown that nikethamide alone decreased mor-
tality when injected 90 min. prior to EPN. Niketh-
amide plus atropine immediately following EPN
only slightly increased survival time of female
mice; nikethamide alone or combined with atro-
pine more effectively protected female guinea
pigs. Nikethamide plus atropine immediately
following TEPP, OMPA, Parathion or DFP in
female mice or rats or physostigmine in rats did
not alter the toxicity of these agents. EPN poi-
soned rats and mice which only partially degrade
nicotinamide, a metabolite of nikethamide, were
protected by 1 gm/kg of nicotinamide. Guinea
pigs, which completely degrade nicotinamide,
were not protected. Nikethamide or nicotinamide,
with or without atropine, injected repeatedly in
female rats over a period of 8 hr. prior to TEPP
did not alter its toxicity. Nicotinamide exerted
some protection against Parathion and OMPA in
female rats. Tryptophan and nicotinic acid pro-
tected against EPN poisoning in female rats.
N’-methyl nicotinamide (NMN) exerted little
or no protection against EPN poisoning in female
rats alone or when combined with the dye cyanine
863, which inhibits tubular excretion of NMN
(J. Pharm. and Exper. Therap. 113; 148, 1955).
Neither methionine nor dimethylethanolamine
altered the toxicity of EPN and methionine did
not overcome the beneficial action of nicotin-
amide. Ethionine prolonged survival time. Nik-
ethamide and nicotinamide provided 30-100%
protection against in vivo EPN inhibition of cho-
linesterase in rat brain, spinal cord, heart, lung,
diaphragm, intercostal muscle, plasma and red
blood cell.

1548. Methyleellulose as a wetting agent in
blood clot. Morris Rosenretp. Dept. of
Pharmacology and Exptl. Therapeutics, Johns
Hopkins Univ. School of Medicine, Baltimore,
Mad. oe
Methyleellulose alters some of the physical

properties of human blood plasma clot in a direc-

tion that might be favorable for hemostasis. The
clot surface takes on hydrophilic properties in
contrast to its normal hydrophobic character.

This increased wettability facilitates better

spread and adherence of superimposed layers of

clot. Methylcellulose also behaves as a surface
active agent in fluid plasma, elevating the surface
tension from 52 to 72 dynes/em when present in

0.1% concentration (Methocel, Dow). There was

a slight but definite promotion of clot retraction

in platelet poor plasma. No effect on clot wettabil-

ity or on surface tension was observed with other

FEDERATION PROCEEDINGS

Volume 16

hydrophilic colloids; namely, gelatine, dextran,
polyvinylpyrrolidone and carboxymethylcellulose,
When injected intravenously in dogs, methyl-
cellulose entered into a volume of distribution of
about 6% of body weight, which indicates restrie-
tion of the agent to the blood plasma. Blood levels
adequate for wetting action were maintained for
12-24 hr. after a single intravenous injection.
(Supported by a research grant, C-1841, from the
Natl. Cancer Inst. of the Natl. Insts. of Health,
PHS.)

1549. Protective effect of ganglionic blocking
agents on traumatic shock in the rat.
CHARLES A. Ross AND STEPHEN A. HERczEG
(introduced by Karu H. Bryer). Pharmacology
Section, Sharp & Dohme, Div. of Merck & Co.,
Inc., West Point, Pa.

Adrenergic blocking agents have been reported
to reduce effectively the mortality associated
with drum shock in the rat. That pretreatment
with ganglionic blocking agents also significantly
reduces the mortality resulting from total body
trauma in the rat has been demonstrated in the
studies to be presented. The intraperitoneal
pretreatment with appropriate dosages of me-
camylamine, chlorisondamine, pentolinium and
hexamethonium produced significant protection
from the mortality following exposure to drum
shock. Tetraethylammonium in comparable ex-
periments failed to provide significant protection.
These studies provide additional support for the
observation that blockade of the sympathetic
division of the autonomic nervous system during
physical assault prevents the pathologic events
resulting in death.

1550. Epinephrine arteriopathy and thyroid-
ectomy. A. P. Roszkowski* anp Y. T. OESTER.
Dept. of Pharmacology and Exptl. Therapeutics,
Stritch School of Medicine and the Grad. School,
Loyola Univ., Chicago, Til.

A severe type of aortic degenerative arterio-
sclerosis is induced in rabbits by means of re-
peated daily injections of large doses of epineph-
rine (JosuB, 1904) (FREIDMANN et al. 1955). This
type of lesion has been observed in the aorta and
primarily involves a degeneration and necrosis
of the elastic and muscular constituents of the
tunica media. Friedmann et al. (1955) were able
to display a 50% incidence of this type of medial
sclerosis when epinephrine alone was injected
for 15 days or less. Oester and Mikulicich (1951)
showed that exogenous thyroxine greatly aug-
ments this sclerogenic effect of epinephrine and a
high sclerogenic incidence (89.5%) was reported
when thyroxine was introduced along with epi-
nephrine. It had been reported by Lortat and
Sabareanu (1904) that thyroidectomy suppressed
epinephrine sclerosis. This finding has been

oh Oak ee ee ee Cee ee Om ee, ie ee ee ee

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RCZEG
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ESTER.
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March 1956

questioned by many. Because of these findings it
was decided to examine further the role the thy-
roid plays in the induction of this type of arterio-
sclerosis. A group of 10 rabbits were thyroid-
ectomized. A period of 7 days was allowed for
recovery. Following this period, each animal
received intravenous injections of epinephrine,
50 ug/kg daily, for a total of 15 injections (the
usual epinephrine sclerogenic regimen). In ‘an
endeavor to determine how complete the thyroid-
ectomy had been, 5 ue of I!%! was administered
subcutaneously to each rabbit, 24 hr. before being
killed. The thoracic aorta and heart were removed
and examined for aortic lesions. The trachea and
adjacent areas, where normally thyroid tissue is
found, were removed. In addition, an indifferent
tissue, namely, muscle from the thigh, was taken
as a control. The thyroid area and thigh muscle
were digested separately and the radioactive
uptake of each was measured. Upon autopsy
examination of the thyroidectomized animals, no
evidence of aortic sclerosis was displayed, nor
was there any evidence of other aortic lesions.
This completely negative result contrasts sharply
with the finding of a 50% incidence of aortic
sclerosis in nonthyroidectomized rabbits. A rela-
tively small radioactive count was obtained from
the tissue in the thyroid area, in 8 of the 10 thy-
roidectomized animals. This would indicate that
some thyroid remnants were still functioning in
this area. The medial sclerosis produced by large
intravenous injections of epinephrine is not pro-
duced in thyroidectomized rabbits.

1551. Gastric antisecretory and antimotility
agents. F. E. Rotu,* E. Ecxuarpt,* A.
Makovsky* ano W. M. Govier. Research Div.,
Schering Corp., Bloomfield, N. J.

The current availability of a multitude of anti-
cholinergic drugs for the management of peptic
ulcer has served to reemphasize the clinical
requirement for an antisecretory agent with pro-
longed oral efficacy and without development
of tolerance or disagreeable side effects. After
preliminary studies with several series of com-
pounds, N-methyl-4-piperidyl benzilate methyl
iodide (Sch 3444) was selected for clinical trial.
It was as effective as Pamine in inhibiting gastric
secretion in the Shay rat at oral doses of 1, 2 and
5 mg/kg. It was of interest to observe that an
analogue of Sch 3444, N-ethyl-3-piperidyl ben-
zilate methobromide (Piptal), did not possess
antisecretory action in our rat studies. In the
dog, Sch 3444 (0.5 mg/kg, orally) appeared to be
twice as effective as Pamine or Piptal (1.0 mg/kg)
in the inhibition of gastric emptying time follow-
ing a barium meal. However, Sch 3444 and Piptal
did not produce mydriasis in these studies while
Pamine did. No signs of central nervous stimula-
tion were noted in rats (10 mg/kg, orally) or mice

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

477

(1 mg/kg, intraperitoneally) with the above
compounds. Mydriasis was observed in both the
rat and mouse after Pamine but not after Sch
3444. Sch 3444 demonstrated somewhat less anti-
cholinergic potency than did Pamine (guinea pig
intestinal strip, mouse antisialogogue and dog
blood pressure tests). Acute and chronic toxicity
studies with Sch 3444 in rats and mice have indi-
cated a good safety margin and no signs of cumu-
lative action.

1552. Activity relationships in animals of a
series of organic di-azides. L. W. RotH aNp
B. B. Morputs.* Dept. of Pharmacology, Abbott
Labs., N. Chicago, Il.

Six straight-chain di-azides (synthesized in the
Organic Chemistry Dept.) having the general
structure N;-(CHe)n-Nz were tested for various
pharmacological characteristics. All were found to
produce a fall of blood pressure, both in normoten-
sive and hypertensive animals, by i.v., oral or
i.m. administration. Duration and magnitude of
response were found to be related to chain length,
with a maximal peak at C; and a decline to either
side of this most potent member of the series.
Acute toxicity tests by various routes in mice and
other animals showed a variability which did not
correlate well with the vasodilator activity. One
representative (C;) was chosen for more extensive
study. In normal unanesthetized dogs, high doses
produced depression, emesis and death, with no
consulvant symptoms. At lower doses it caused a
significant lowering of blood pressure, measured
by direct arterial puncture. The hypotension had
a duration of several hours after intramuscular
administration. The predominantly peripheral
site of action was verified by a variety of tests,
including Nolf preparations, administration of
antihistaminics, ganglionic or adrenergic blocking
agents, elicitation of vasomotor reflexes, decere-
bration, pithing, etc. Reactivity to sympa-
thomimetic amines was relatively unchanged.
Langendorff and Starling heart preparations were
used to study cardiac and coronary effects and
electrocardiographs on anesthetized animals dur-
ing the injection of azides, to observe changes in
pattern or rate.

1553. Prevention of ventricular fibrillation by
a somatotrophic hormone preparation in
experimental coronary artery occlusion.
Sot RotHMAN AND Davip H. Warxns (intro-
duced by GrorG CRoNHEIM). Research Div.,
Riker Labs., Los Angeles, Calif., and Univ. of
Colorado, Med. Ctr., Denver.

Laborit has reported that somatotrophic hor-
mone prevented ventricular fibrillation due to
coronary artery occlusion. Two types of experi-
ments were performed in mongrel dogs: occlusion
of either the anterior descending or circumflex

478

branch of the left coronary artery in ‘open chest’
dogs; or ligation of the circumflex artery in a
chronic preparation. The somatotrophic hormone
used was a semipurified material prepared by
Choay Labs., France. One Choay Unit when as-
sayed in ‘plateaued’ female rats approximated
0.2-0.5 Evans Units. Test dogs were given 50 or
100 vu of somatotrophic hormone i.v. or i.m. at
16, 4 and at 0 hrs. before surgery. Control animals
received saline injections. All experimental
groups were controlled. In some of the chronic
studies the animals were observed for 14 days
and then killed. In ‘open chest’ experiments sur-
vival at 30 min. was 4/12 for the control (C) and
7/11 for the test (T) group; at 60 min.: C = 2/12,
T = 6/11. In ‘closed chest’ preparations survival
values were: 1 hr.: C = 15/28, T = 25/32; 24 hr.:
C = 10/28, T = 20/31; and in a 14-day study:
C = 6/17, T = 11/20. Protection by the hormone
preparation was seen in all experiments. When
the results were subjected to statistical analysis,
using survival at 30 min., 1 hr., 24 hr. and 14 days,
protection was found to be highly significant
(P < 0.001).

1554. Effectiveness of protection with chlor-
promazine administered (A) during shock
and (B) in combination with varying sup-
portive therapy. E. A. RoveNsTINE AND S. G.
Hersuey.* New York Univ. Postgrad. Med.
School, New York City.

Rats pretreated with chlorpromazine (2.5 mg/
100 gm b. wt.) were subjected to 3 hr. of hemor-
rhagic hypotension (50 mm Hg) via a self regulat-
ing bleed-out reinfusion reservoir. After this
period, blood remaining in the reservoir was rein-
fused and the animals observed for 48 hr. for sur-
vival. These animals showed an 89% survival
rate as compared with 40% in animals not given
chlorpromazine. When chlorpromazine (0.6 mg/
100 gm b. wt.) was administered intra-arterially
after the 1st hr. of hypotension the survival rate
was 33%. When this dose was given at the end of
each hour of the 3-hr. hypotensive period the
animals showed a 40% survival. Rats receiving
this dose at*the end of the 3rd hr. had a 42% sur-
vival. A number of other dose ranges and timing
schedules were also studied. These observations
indicated that rats receiving chlorpromazine after
the onset of hemorrhagic hypotension were not
significantly protected. In another series of ex-
periments chlorpromazine was administered prior
to hemorrhage as in the controls except that the
final reinfusion of whole blood was omitted. None
survived. In a further modification, gelatin was
administered as a blood replacement in volume
equal to the blood remaining in the reservoir.
None survived. One set of animals which received
a suspension of their own red cells in gelatin after
a period of hemorrhagic hypotension, showed 43%,

FEDERATION PROCEEDINGS

Volume 16

survival. Pretreated animals, not given whole
blood, were not appreciably protected. The results
indicate that chlorpromazine-induced protection
against irreversible hemorrhagic stress is de-
pendent upon pretreatment and whole blood
supportive therapy.

1555. Cardiovascular effects of norepinephrine
in the dog. Rospert L. RussELL AND JAMES E,
RANDALL (introduced by B. A. WEsTFALL).
Dept. of Physiology and Pharmacology, Univ. of
Missouri, Columbia.

Recent literature has indicated that clinical
doses of norepinephrine will elicit electrocar-
diographic changes and that a vasopressor drug,
N-methylphenyl-tertiary-butylamine sulfate, may
exhibit tachyphylaxis. The present investigation
was undertaken to study the effects of large doses
of norepinephrine upon the dog. Arterial pressure
and limb-lead electrocardiograms were taken
simultaneously in 9 anesthetized dogs. Norepi-
nephrine (Levophed) with concentration of 0.8
mg/ml in isotonic saline was infused intravenously
at a rate sufficient to maintain arterial systolic
pressure at or above 250 mm Hg. Tachyphylaxis
was observed by noting infusion rate and failure
to maintain a pressure of 250 mm Hg. Four ani-
mals exhibited tachyphylaxis after 30 min. of
infusion, 4 exhibited marked tachyphylaxis after
60 min. of infusion, and 1 showed slight tachy-
phylaxis after 60 min. of infusion. Total dosage
per animal ranged from 1.0 to 10.5 mg/kg. All
electrocardiographic records showed marked pre-
dominance of beats originating from ventricular
foci following initiation of the norepinephrine
infusion. These rather high doses of norepineph-
rine were noted to increase the heart rate. In
general, the electrocardiograph resumed a normal
pattern during the infusion period, exhibiting
ectopic beats when the infusion rate was increased
too rapidly. (Levophed courtesy of Wintrhop-
Stearns, Inc.)

1556. Rate of recovery of rats and mice exposed
to x-rays. Paut R. SALERNO. Atomic Energy
Med. Research Project, Western Reserve Univ.,
Cleveland, Ohio.

Male Carworth rats and CF, female mice were
exposed to single doses of X-rays ranging from 130r
to 520 r and at intervals of 1-18 days later the
sensitivity to a 2nd exposure of X-rays was com-
pared with that of control animals. The residual
cumulative effect from the previous radiation dose
was assessed from the mortality data and parallel
studies of the peripheral blood. Mice appear to
recover at a significantly more rapid rate than
rats, the difference in residual dose being most
prominent during the period 4-8 days after a Ist
exposure of 390 r. In our laboratory the LDs for
mice and rats is 585 r and 660 r, respectively.

| i — oe)

=
So =

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stolic
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ormal
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March 1956

Although the blood leukocyte count of rats ex-
posed to'390 r was reduced to less than 40% of
normal at 12 days, a 2nd dose of 260 r resulted in
hematological depression and recovery which
were not significantly different from that produced
by a single dose of 260 r in control rats. In 2 groups
of rats exposed to a single dose of either 260 r or
390 r, the peripheral white cell count rose from a
level of 3000 to 6000 cells/mm? in the periods of
day & to 11 and day 8 to 14, respectively. During
this process of active recovery, the sensitivity to
a subsequent midlethal dose of x-rays becomes
comparable with that of control animals. The
LD» Of both rats and mice determined 12 days
after an initial dose of 390 r or less was not sig-
nificantly different from that of normal animals.

1557. Influence of hesperidin methyl chalcone
on experimental steroid hypertension. E.
SaLGapo* aNp D. M. Green. Nepera Chemical
Co., Yonkers, N. Y.

Hesperidin methyl chalcone (HMC) was re-
ported by Rinehart to prevent the hypertension
and tissue lesions produced by DCA in salt-treated
rats (Ann. N. Y. Acad. Sct. 61: 693, 1955). In a
repetition of this experiment, male Sprague-
Dawley rats were unilaterally nephrectomized
and given 0.87% saline to drink. Three 25-mg DCA
pellets were implanted subcutaneously in 20 ani-
mals, 10 of which also received, subcutaneously,
20 mg of HMC twice daily. The experiment was
terminated after 40 days. Both groups showed
comparable rises in fluid intake (ml/gm/day:
DCA-treated, 0.57; DCA-HMC-treated, 0.58;
controls, 0.29) and blood pressure (mm Hg: DCA-
treated, 196; DCA-HMC-treated, 201; controls,
131). However, the incidence and severity of
lesions in kidneys, brain and arteries were sig-
nificantly and materially reduced in the HMC-
treated animals. Apparently, tissue lesions pro-
duced by DCA can be dissociated from
hypertension.

1558. Effects of Pitressin-DCA treatment in
hypophysectomized rats. E. SALGADo* AND
D. M. Green. Nepera Chemical Co., Yonkers,
NW, Y.

We have previously reported the failure of DCA
to induce hypertension in saline-fed, hypophysec-
tomized rats, although fluid exchange is increased.
Simultaneous treatment with various anterior
pituitary preparations did not overcome this
failure. In present experiments, the effects of
posterior pituitary extract were studied in groups
of unilaterally nephrectomized, hypophysecto-
mized rats, given 0.87% saline to drink. One group
was injected with Pitressin tannate, 0.5 u/rat/day,
subcutaneously; a 2nd was implanted with three
25-mg DCA pellets; while a 3rd received both
treatments. A 4th group served as a control. The

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

479

experiment was terminated after 49 days. Both
the DCA-treated and the DCA-Pitressin-treated
groups exhibited a comparable rise in fluid intake,
to about double the control value. Both showed,
also, a small increase in blood pressure relative to
the controls, but not to hypertensive levels. The
group treated with Pitressin alone did not differ
from the controls in fluid intake or in blood pres-
sure. At autopsy, none of the animals showed
lesions attributable to the drug treatments. All
hypophysectomies were complete. Apparently,
posterior, like anterior pituitary extracts are
inadequate to replace the living gland in permit-
ting DCA-induced hypertension.

1559. Methemoglobin formation in the cat in
relation to treatment of cyanide intexica-
tion. J. PALMER SAUNDERS* AND S. RicHAakp
Hetsey* (introduced by R. V. Brown). Pharma-
cology Branch, Chemical Corps Med. Labs., Army
Chemical Ctr., Md.

Sodium nitrite and p-aminopropiophenone
(PAPP) are considered to be useful in the treat-
ment of cyanide poisoning because of their ability
to produce methemoglobinemia. Methemoglobin
(MetHb) combines with cyanide to form cyan-
methemoglobin (CNMetHb). In a study of the
formation of MetHb by i.v. administration of
nitrite or PAPP in the cat, it was found that after
the administration of nitrite MetHb reached max-
imal values within 5 min. of 0.6, 1.0 and 1.8 gm/100
ml blood for nitrite doses of 5, 7.5 and 10 mg/kg,
respectively. After these maximal values had been
reached, the MetHb levels remained relatively
constant over a period of 4 hr. With 15 and 25
mg/kg doses of nitrite, the MetHb levels rose
rapidly and reached maximal values of 4 and 8 gm
MetHb/100 ml blood in 60 and 120 min., respec-
tively. In the case of PAPP, MetHb again formed
rapidly, reaching maximal values within 5 min.
of 1.5, 4.2 and 4.8 gm/100 ml blood for doses of 0.5,
1.0 and 2.0 mg/kg. After the maximal values had
been reached, the MetHb levels declined slowly
over a period of 4 hr. Based on the fact that 1 gm
MetHb requires approximately 2.9 mg cyanide for
complete formation of CNMetHb, it can be as-
sumed from the experimental data that it is
necessary to give at least 10 mg/kg sodium nitrite
or 0.5 mg/kg PAPP in order to expect recovery
from the acute administration of 2 LDims (3.5
mg/kg) sodium cyanide to cats. Experiments
reported elsewhere in these Proceedings support
these assumptions.

1560. Chemical estimation of acyl glucuro-
nides: formation and urinary excretion in
the human. Davip ScHacuTER (introduced by
Herman I. Cuinn). Dept. of Pharmacology and
Biochemistry, School of Aviation Medicine,
USAF, Randolph AFB, Randolph Field, Tez.

480

Acyl glucuronides, in which an acyl group is
linked to p-glucuronic acid, differ from other glu-
curonides (phenolic and alcoholic) by their ready
hydrolysis in alkaline solution. The present com-
munication describes other unique properties, as
studied with crystalline benzoyl glucuronide and
the acyl glucuronides which appear in human urine
after ingestion of benzoate and salicylate. Ben-
zoyl glucuronide is converted quantitatively to
benzoyl hydroxamate under the conditions de-
scribed by Lipmann and Tuttle (J. Biol. Chem.
159: 21, 1945) for a similar conversion of high-
energy acyl phosphates. This affords a sensitive
procedure for its estimation. In a normal man oral
doses as low as 1.0 gm of benzoate were followed
by urinary excretions of detectabie benzoyl glu-
curonide, further identified by demonstrating:
1) identity of its Rr value with that of crystalline
benzoyl glucuronide on paper chromatograms, 2)
identity of the Rr value of its hydroxamate with
benzoyl hydroxamate on paper chromatograms,
and $) its ready hydrolysis by bacterial B-glucu-
ronidase (Sigma). The urinary excretion curves of
benzoyl glucuronide and hippurate following
varying doses of benzoate in man will be pre-
sented. In a normal human, ingestion of salicylate
caused the urinary excretion of salicyl acyl glu-
curonide, salicyl phenolic glucuronide and salicyl-
urate. Specific methods have been developed for
the estimation of each of these metabolites, and
their urinary excretion curves following various
doses of salicylate will be demonstrated.

1561. Absorption of drugs by the rat intestine.
Lewis S. ScHANKER,* C. Aprran M. Hoapen,*
ParKHuRST A. SHORE* AND BERNARD B. BropieE.
Labs. of Chemical Pharmacology and Kidney and
Electrolyte Metabolism, Natl. Heart Inst.,
Bethesda, Md.

Intestinal absorption of a number of organic
bases and acids has been studied using the small
intestine in situ of the anesthetized rat. A 1.0 mm
solution of drug in isotonic saline solution (pH 7.2)
at 37°C was perfused through the entire length of
small intestine at a rate of 1.5 ml/min. Volume
changes were. followed with radioinulin. Addi-
tional information was provided by terminal
plasma concentrations and drug ultrafiltrability
in intestinal fluid. Absorption of organic bases ap-
pears to be related to ionization constants. Theo-
phylline, antipyrine, aniline and aminopyrine
(pKa 0.7 to 5.0) were absorbed to the extent of 30-
50% while stronger bases such as quinine, ephed-
rine, Priscoline, Darstine and tetraethylammo-
nium (pKa > 8.0) exhibited absorption values of
only 3-13%. Absorption of organic acids does not
appear to be pKa dependent. Salicylate, benzoate,
p-hydroxypropiophenone and _ phenylbutazone
(pKa 3.0-7.8) were absorbed to the extent of 40-
50%. Only 1 of the acids studied, acetylsalicylic

FEDERATION PROCEEDINGS

Volume 15

acid (pKa 3.5), was relatively poorly absorbed, the
value being 19%. Preliminary results with deu-
terium oxide indicate that it is absorbed to the
extent of about 40%. Factors relevant to very
rapid absorption such as plasma binding and
lumenal mixing will be considered. From the
reported values for splanchnic blood flow, it ap-
pears that the absorption of the rapidly absorbed
drugs (e.g. aniline and salicylate) and D2O may
be blood flow limited.

1562. Effects of dinitrophenol on carbohy-
drate metabolism of rat brain mince.
Hersert S. Scowartz* anp S. B. Barker.
Dept. of Pharmacology, Univ. of Alabama Med,
Ctr., Birmingham.
2,4-Dinitrophenol (DNP) stimulates the in

vitro respiration of rat brain in the presence of

appropriate substrates. A crude mince of rat cere-
bellum and cerebrum is incubated in oxygen at
37°C. in a phosphate-free Krebs-Ringer saline
solution buffered at pH 7.4 with 0.02 m tris-(hy-
droxymethy])-aminomethane. Previously reported
studies (Am. J. Physiol. 171: 765, 1952) indicated
that glucose, mannose, pyruvate and lactate sup-
ported DNP stimulation when both substrate and
DNP were present from the start. Fructose and
some other substrates have since been found to
support a secondary stimulation if the substrate
and tissue are preincubated before the DNP is
added. Although glucose supports a 2-fold in-
crease in respiration with DNP without preincu-
bation, the stimulation is intensified when DNP is
added later. Respiration is inhibited when no
exogenous substrate is present with DNP. Glucose
is unable to stimulate respiration after endogenous
preincubation with DNP. Fructose fails to pro-
tect the tissue from DNP inhibition and delayed
glucose tipping does not elicit a stimulation. The
results of carbohydrate studies with DNP confirm
part of the substrate specificity requirements of
purified brain hexokinase reported by Sols and

Crane (J. Biol. Chem. 210: 581, 1954). (Supported

by a grant from the Smith, Kline and French

Fndn.)

1563. Electroencephalographic patterns dur-
ing first stage N.O anesthesia. JoHn F.
Scuwetss, M. Jack FRUMIN AND Ext GoLpEN-
SOHN (introduced by E. M. Pappsr). Depts. of
Anesthesiology and Neurology, Columbia-Presby-
terian Med. Ctr., New York City.

A Grass Oscillograph with a flat response be-
tween 1 and 40 cps was used to record the elec-
troencephalograms of 30 unselected, unpremedi-
cated anesthetized patients during major surgical
procedures. Anesthesia was induced with an 80%
N.O 20% O2 mixture. A continuous intravenous
infusion of 0.5% succinylcholine was used to ef-
fect endotracheal intubation and to maintain

S 2. ee af fn Sie a a [See (ei (le, ee es a a oe es

—

one tnd

e 16

, the
deu-
. the
very

and

the
5 ap-
rbed
may

ohy-
ince.
‘KER.
Med.

e in
ce of
cere-
an at
aline
-(hy-
orted
cated
sup-
e and
> and
nd to
strate
NP is
d in-
incu-
NP is
n no
ucose
enous
) pro-
layed
. The
nfirm
rts of
3 and
ported
rench

dur-
IN F,
LDEN-
nts. of
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se be-
: elec-
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n 80%
renous
to ef-
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March 1956

apnea and immobility. First stage anesthesia and
artificial respiration were maintained with a non-
rebreathing intermittent positive pressure respi-
rator which delivered a 65% N2O-35% Oz inspira-
tory mixture. The inflating pressure was
servo-controlled to maintain an end-expiratory
CO: concentration of approximately 5%. Amnesia
for the surgical procedures was complete in all
patients. The electroencephalographic changes
noted in the trans-frontal, trans-occipital and
occipito-frontal leads consisted of 1) moderate
voltage depression, 2) a 4-2 cps reduction in alpha
frequency, 3) medium voltage activity at 4-7 cps.
No consistent or significant change in the ampli-
tude or amount of rapid activity (16-28 cps) was
noted. The pre-anesthetic pattern reappeared 2-5
min. following the inhalation of 100% O:2 or room
air.

1564. Cardiovascular actions of Viadril
(21l-hydroxypregnanedione sodium succi-
nate). A. ScRIABINE* AND D. E. HutcHeon.
Research Labs., Chas. Pfizer & Co., Inc., Brooklyn,
7. ee a
Viadril is a new intravenous anesthetic with a

high therapeutic index, (P’an et al. J. Pharmacol.

115: 432, 1955). In contrast to sodium thiopental,

Viadril in anesthetic doses did not cause ven-

tricular extrasystoles or alternating ventricular

rhythms in dogs injected with morphine. In intact
cats and dogs under Viadril anesthesia (100 mg/
kg), epinephrine (10 and 20 ng/kg was observed to
have less tendency to produce ventricular ex-
trasysteles than in animals under sodium thio-
pental anesthesia (25 mg/kg). Viadril in doses of
5-20 mg/kg decreased the response of cat’s nicti-
tating membrane to the preganglionic electrical
stimulation of the sympathetic chain. The drug
had no adrenolytic properties as judged by its
inability to block epinephrine induced blood pres-
sure responses. Viadril was tested for vagolytic
activity in 6 cats under sodium pentobarbital
anesthesia. Viadril 12.5 mg/kg was on the average
20% and 25 mg/kg was 59% effective in abolishing
the cardiac slowing produced by electrical stimu-
lation of the peripheral end of right vagus. In the
dog’s hind limb preparation 2 mg/kg Viadril
intra-arterially was observed to increase the
femoral blood flow recorded by the Wilson ro-
tameter. In isolated perfused cats’ hearts, Viadril
had less depressant activity on the amplitude of
contractions than sodium thiopental. Both agents
increased coronary inflow. In concentrations up
to 6.7 mg/100 ml, Viadril did not depress the force
of contractions of isolated cats’ heart papillary
muscles significantly when 1.3 mg/100 ml of sodium

thiopental produced a definite depression. Only a

slight reduction in excitability of papillary mus-

cles was noted after Viadril in doses as high as

13.3 mg/100 ml.

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

481

1565. Influence of N-ethyl-3-piperidyl di-
phenyl acetate on premature ventricular
contractions. Luoyp D. SzaceR anp Davip
Baumann.* Dept. of Physvology and Pharma-
cology, Univ. of Arkansas School of Medicine,
Little Rock.

We have demonstrated in experimentai animals
that N-ethyl-3-piperidyl-diphenyl acetate pro-
longs the refractory period, decreases the excita-
bility of auricular muscle, is effective in prevent-
ing chloroform-adrenalin induced ventricular
fibrillation and is effective in converting digitalis
induced premature ventricular contractions to
normal sinus rhythm. Eleven patients presenting
premature ventricular contractions have been
treated with the drug intravenously. A 0.55-1.0%
solution in glucose was administered slowly at
first and the rate was increased until disappear-
ance of the arrhythmia or the development of side
actions. In 8 patients, there was a complete
disappearance of the premature ventricular con-
tractions for from 2 to 36 min. The persistence of
the effect after discontinuing the drug was from
1 to 34 min. The rate of administration of the drug
required to stop the premature contractions
varied from 0.16 mg/kg/min. to 0.69 mg/kg/min.
An additional patient showed a decrease in the
frequency of premature contractions. Six patients
showed an increase in blood pressure and 4 an
increase in heart rate during the administration
of the drug. The only ECG change other than
disappearance of the arrhythmia was a flattening
of T wave in 1 subject.

1566. Relation of clearing factor inhibitors to
hyperlipemia. JosepH SEIFTER AND Davin H.
Barper*. Wyeth Inst. for Med. Research, Radnor,
Pa.

Protamine sulfate, sodium cholate, diisopro-
pylfluorophosphate (DFP) and the dialyzate of
plasma from cortisonized animals have been re-
ported to prevent the delactescent action of
heparin clearing factor in vitro. Protamine sulfate
and LM (lipid mobilizer) contained in the dialy-
zate have also been reported to produce hyper-
lipemia in rats and humans following intravenous
administration. Neither sodium cholate nor DFP
was effective when administered to rats or dogs
in nonconvulsant doses, suggesting that the
hyperlipemia was due to stress and not to anti-
heparin or antilipoprotein-lipase activity. Further
evidence for this possibility was obtained in
hypophysectomized rats. These still responded
to LM but failed to develop hyperlipemia after
administration of protamine sulfate and failed to
produce LM after cortisone.

1567. Activity of Ro 2-5383 in comparison with
cortisone, sodium salicylate and phenyl-
butazone on suppression of inflammatory

482

reactions. JoserpH J. Sevitro* aNnp LOWELL

O. RanpDAtu. Dept. of Pharmacology, Hoffmann-

La Roche Inc., Nutley, N. J.

Activity has been found for Ro 2-5383, (prob-
ably 1,3,6-Trimethy] 8,8-diphenyl-1,2,3,4,5,6,
7,8-octahydropyrido [4,3-d]pyrimidine tartrate)
in reducing inflamed tissue with a potency of
about one-half the activity of cortisone, 4 times
the activity of phenylbutazone and 10 times the
activity of sodium salicylate. The anti-edema
activity was measured by the method of Selitto
and Randall (Federation Proc. 13: 403, 1954) using
Brewer’s yeast as the edema inducing agent. Ro
2-5383 and cortisone reduced the size of the
edematous feet of rats to near normal within 24
hr. and maintained activity during 6 days of
treatment. With sodium salicylate and phenyl-
butazone the edema was reduced within a few
hours, remained small for the first 2 days but
increased again by the 3rd day of treatment.
Ro 2-5383 and cortisone were equally active in
inhibiting granuloma deposition on implanted
cotton pellets in rats (MEIER, SCHULLER AND
Desau._zs, Experientia 6: 12, 1950) while phenyl-
butazone was one-half as potent as cortisone and
salicylate was inactive. All 4 compounds were
active in reducing the elevated temperatures of
inflamed rats’ feet. Ro 2-5383 was slightly more
active than cortisone, twice as active as phenyl-
butazone and 10 times as active as salicylate. A
slight hypertrophy of the adrenals with a corres-
ponding slight atrophy of the thymus was pro-
duced by 7 days administration of Ro 2-5383,
phenylbutazone and salicylate to intact rats. No
thymolitic activity was observed when Ro 2-5383
was injected into adrenalectomized rats.

1568. Sex variation in human plasma cholin-
esterase activity. SypNEY P. SHaNor,* Nora
Baart,* GerTRUDE R. van HEEs,* Ervin G.
Erpés* anp Francis F. Foupes. Dept. of
Anesthesiology, Mercy Hosp., and Section on
Anesthesiology, Dept. of Surgery, Univ. of Pitts-
burgh, Pittsburgh, Pa.

A statistically significant sex variation has
been previdusly reported (Davis, et al. Federation
Proc. 12: 315, 1953) in the hydrolysis rate of pro-
caine-HCl (proc.) in human plasma. In the pres-
ent study the enzymatic hydrolysis rate of acetyl-
choline-Cl (ACh), butyrylcholine-Cl (BuCh),
benzoylcholine-Cl (BzCh), succinyldicholine di-
chloride (SDCh), and proc. was observed in freshly
obtained heparinized plasmas taken from 50
young, healthy adults equally divided between
the 2 sexes. In 14 males and 14 females of the above
group the red cell cholinesterase (RChE) activity
with ACh and acetyl-8-methylcholine-Cl (MeCh)
was also observed. With the exception of proc. the
hydrolysis of all substrates was measured with
Warburg’s manometric method. The hydrolysis

FEDERATION PROCEEDINGS

Volume 15

of proc. was observed with an u.v. spectropho-
tometric method (Katow, J. Pharmacol. & Exper.
Therap. 104: 122, 1952). The substrate concentra-
tions with plasma cholinesterase (PChE) was
2.2 X 10?% m and with RChE 3 X 10-3 m. The
substrate concentration of proc. was 5 X 107° m.
All experiments were done at pH 7.4 and 37°C,
There was a statistically significant sex variation
in the hydrolysis rates of all the substrates hy-
drolyzed by PChE. The activity of the female
plasmas ranged from 64% to 74% of that of the
male plasmas, with ¢-values ranging from 4.5 to
5.0. No sex variation was observed in the activity
of RChE.

1569. Effects of cortisone on the perfused
rabbit heart. Marvin SHELTON,* WALTER M.
Booker, ADELEKE ADEYEMO* AND GILBERT
AuLeN.* Dept. of Pharmacology, Howard Univ.
Med. School, Washington, D. C.

In a previous report we have shown that cor-
tisone causes a decrease in ventricular stroke and
subsequent failure of the heart. It was shown in
the report that the heart was more sensitive to the
‘cortisone effect’ when it was perfused with fluid
low in potassium and was less sensitive when per-
fused with fluid high in potassium. Also it was
suggested in the study that the effect of cortisone
might be quantitative. In the present study we
have undertaken to correlate the dose-time-re-
sponse in order to provide more information on
the quantitative effect of cortisone. Doses rang-
ing from 0.25 mg to 2.5 mg were added to the
perfusate at fixed time intervals of 3 min. The
data show clearly that the response of the prep-
arations in time is based on concentration; that
is, the more dilute the solution of cortisone the
longer is required for the ‘cortisone effect’ to
appear. These dose-time-response data will be
tested on solutions of variable potassium concen-
trations. Also in this report data will be presented
comparing some other adreno-steroids with
cortisone.

1570. Effect of ambient temperature on ther-
mal responses to drugs. IRvinGc SHEMANO*
AND Mark Nickerson. Dept. of Physiology and
Med. Research, Univ. of Manitoba Faculty of
Medicine, Winnipeg, Canada.

Unanesthetized female rats (150-200 gm) were
placed in individual wire mesh holders in a con-
stant temperature room and their colonic tempera-
tures recorded with constantan-copper thermo-
couples inserted 5-6 em beyond the anal sphincter.
After an equilibration period of 1 hr., the agent
under study was injected subcutaneously and
temperatures were recorded hourly up to 3 hr.
after injection. Each rat was used as its own con-
trol during separate, equal periods at the same
ambient temperature. Chlorpromazine (25 mg/kg)

anc
the

inj

so f.
9 de

2 15

yho-
per.
tra-
was
The
5M,
a;
tion
hy-
nale
the
D to
vity

ised

sERT

Iniv,

cor-
and
n in
» the
fluid
per-

sone
y we
p-Te-
n on
ang-
the
The
yrep-
that
. the
to
1 be
\cen-
nted
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her-
ANO*
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were
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con-
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g/kg)

March 1956

elicited a, consistent hypothermia at all ambient
temperatures studied up to 33°C. Dinitrophenol
(20 mg/kg) produced a consistent hyperthermia
above a ‘critical’ ambient temperature range of
20 to 22°C and a consistent hypothermia at lower
temperatures. The ‘critical’ ambient temperature
for Hydergine (1.0 mg/kg), ergotamine (5.0
mg/kg), lysergic acid diethylamide (LSDA)
(1.0 mg/kg) and 5-hydroxytryptamine (serotonin)
(8.0 mg/kg) was about 30°C, and for reserpine
(1.0 mg/kg) about 25°C. These results indicate
that ambient temperature is a critical factor in
evaluating thermal responses to drugs and suggest
that many such responses involve some inter-
ference with central body temperature regulating
mechanisms. Even dinitrophenol, which has been
assumed to have a predominantly peripheral ef-
fect increasing heat production, appears to follow
this pattern of action.

1571. Methods of analysis of chlorisondamine
chloride in body fluids. HERBERT SHEPPARD,*
A. J. PLUMMER AND Nancy D. SaBBaau.*
Research Dept., Ciba Pharmaceutical Products,
Inc. Summit, N. J.

A method was developed for the analysis in
body fluids of chlorisondamine (Ecolid™) chlo-
ride, 4,5,6,7-tetrachloro-2-(2-dimethylamino-
ethyl)-isoindoline dimethochloride, a bisquater-
nary compound showing prolonged ganglionic
blocking activity. The analysis was based on the
extraction with chloroform of a complex of chlo-
risondamine with brom-cresol-green and _ the
reading of the yellow color at 420 mu. Preparation
of the solution for analysis varied with the tissues
or fluid under study. Blood plasma was simply
dialyzed against 9 volumes of buffer and the dialy-
sate used for analysis. Urine was passed through a
column of cation exchange resin and the chlo-
risondamine bound by the resin was eluted with
2N NaCl. The NaCl solutions obtained in this
manner were then reacted with the dye. Urinary
excretion and blood levels of chlorisondamine in
dogs were determined after intravenous injection
and the results indicated considerable uptake of
the drug with relatively slow release. Urinary
excretion in 24 hr. was approximately 73% of the
injected dose. Paper chromatograms of the ex-
creted material indicated that the chlorisonda-
mine was unchanged.

1572. Blood flow in the thyroid gland of the
dog. JAMES H. SHINABERGER* AND H. D.
Bruner. Dept. of Physiology, Emory Univ.,
Emory University, Ga.

The thyroid gland is accepted as having the
highest blood flow per unit weight of any mam-
malian tissue, a value of 560 cc/100 gm/min. This,
so far as can be determined, is quoted from data on
9 dogs published by Tschuewsky in 1903 using the

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

483

Ludwig stromuhr. Study of the thyroid artery in
25 dogs showed 3-4 fairly regular extrathyroidal
branches which could be ligated and 1, the sterno-
hyoid branch, could be cannulated. Accordingly,
a small caliber bubble-flowmeter was connected
from the femoral into this branch and when flow
was established, the thyroid trunk and all extra-
thyroidal branches were carefully ligated without
disturbing those to the gland. In 15 anatomically
and otherwise satisfactory experiments, the aver-
age flow was 4.8 cc per unilateral gland per min. or
697 cce/100 gm of thyroid per min. The standard
deviations of both values were large; the coeffi-
cient of variation for the flow per gram was 54.5%
and 60.5% for the flow/100 gm of thyroid. The
flows gradually decreased with time although no
evidences of clotting were found in the circuit.
Nor-adrenalin and /-adrenalin acted as vasocon-
strictors and acetylcholine as a vasodilator;
atropine and tetraethylamine were without effect
(6 dogs). Vagotomy at various levels and section
of the recurrent laryngeal nerve invariably were
followed by increased peripheral resistance in the
gland (6 exps.), but stimulation of these severed
nerves also gave increased peripheral resistance.
Additional experiments are planned. (Supported in
part by a Lederle Fellowship and in part by a Life
Insurance Med. Research grant).

1573. Mechanism of serotonin-release by
reserpine. PARKHURST A. SHORE,* ARVID
CaRLSSON* AND BERNARD B. Bronte. Lab. of
Chemical Pharmacology, Natl. Heart Inst.,
Bethesda, Md.

It has been shown that reserpine administration
to animals causes liberation of serotonin from its
various body depots. This release occurs in brain,
intestine and blood platelets. A convenient system
for study of in vitro release consists of rabbit blood
platelets suspended in plasma and incubated with
reserpine at 37°C in an atmosphere of nitrogen. It
has been found that a very low concentration of
reserpine, 0.3 y/ml (5 X 10-7 m) liberates serotonin
maximally from platelets. It can be calculated
that 1 molecule of reserpine liberates a large num-
ber of serotonin molecules indicating that the
release of serotonin is not a simple displacement
by reserpine. Of a large number of substances
studied, including other alkaloids of Rauwolfia,
only the pharmacologically active Rauwolfia alka-
loids, reserpine, rescinnamine and 11-desmethoxy-
reserpine effect the release. This is in agreement
with in vivo experiments in which only the
‘tranquillizing’ alkaloids cause serotonin release
from brain. Since neither histamine nor protein is
liberated from platelets, no general change in
permeability is involved. No release of serotonin
occurs when the incubation is carried out at 0°C.

484

1574. Anticonvulsive actions of bicyclohep-
tene derivatives in mice. E. A. Sregmunp,*
R. A. Capmus,* A. H. CampsBEtt, Jr.,* M. J.
PENEK* AND Go Lv. Johnson & Johnson Research
Fndn. and Research Div. of Ethicon, Inc., New
Brunswick, N. J.

Compounds of 3 series (carbonylurea, alkylurea
and acidamide) of bicycloheptene derivatives were
administered orally in gum tragacanth to Car-
worth Farms male mice. The anticonvulsive
actions were determined by both the Maximal
Electroshock Seizures (M.E.S.) Method and by
the Subcutaneous Pentylenetetrazol (Metrazol)
Seizures Method. The respective Median Effective
Doses (ED50’s), Median Minimal Neurotoxic Doses
(TDs50-s), and Median Lethal Doses (LD50.5) were
determined, and the corresponding Protective
Indices (P.I.’s) and Therapeutic Indices (T.I.’s)
were calculated for each compound. Comparative
data were obtained for several reference stand-
ards. 2-Exomethylbicyclo-(2,2,1)-5-heptene-2-
carbonylurea (ERL-286) was found to be the most
potent compound in all 3 series, having an ED5
(by the M.E.S. test) of 75 mg/kg (0.39 mm/kg) and
Aan EDs (by the Metrazol test) of 31 mg/kg (0.16
mM/kg). It is more potent than its cyclohexenyl
analog, though with smaller P.I.’s and T.I.’s. In
potency against Metrazol-induced seizures, it
approaches the range of phenobarbital sodium.
Similarly, it is much more potent than trimetha-
dione and paramethadione and more potent than
phenacemide. The P.I.’s and T.I.’s compare favor-
ably with those of these 3 drugs. Bicyclo-(2,2,1)-
5-heptene-2-endocarbonylurea (ERL-227) is mod-
erately effective in combating both electrical and
chemical seizures, whereas its cyclohexeny] analog
is inactive at 1000 mg/kg. Anticonvulsive actions
vs. Metrazol seizures and/or vs. M.E.S. were also
observed in the alkylurea and acidamide series,
though being comparatively less potent.

1575. Modern concepts of thymic physiology.
VaucHAN P. Srmmons (introduced by H.
Beckman). Dept. of Pharmacology, Marquette
Univ. School of Medicine, Milwaukee, Wisc.
Evidencé ‘is presented that the long-revered

notions regarding the virtual disappearance of

the thymus in adulthood are purely mythical.

Thymus specimens from adult human beings and

commonly used laboratory animals are demon-

strated. Sequential changes in the guinea pig
thymus during the 16 day estrus cycle are pre-
sented depicting nearly every histologic variation
described for the thymus including myeloid meta-
plasia; all are normally recurring phenomena.

Smallest immediately after ovulation, regenera-

tion of the severely depleted organ occurs very

rapidly at about the time of cessation of corpus
luteum function, apparently as a result of the ac-

FEDERATION PROCEEDINGS

Volume 16

tivity of the large clear cells of the medullary por-
tion and not by an influx of cells from without.
The large thymus seen early in gestation in the
guinea pig is shown to disappear almost com-
pletely as term approaches and at a time when it is
loaded with mucoprotein-laden cells which are
seen leaving the thymus via the lymphatic drain-
age. They are extremely rare in the lymph nodes
but are plentiful, even in early pregnancy, in the
spleen where it appears that they are being filtered
out. The suggestion is made that in human beings
the thymus may be a source of the increased circu-
lating mucoprotein during late pregnancy as well
as in cancer and other conditions associated with
new tissue growth or replacement. Attention is
called to the possible role of the thymus as an im-
portant factor in such diverse conditions as leu-
kemia, thyrotoxicosis, the allergies and hypersen-
sitive states, aplastic anemia and myasthenia
gravis.

1576. Pharmacology of 5-acetylimino-4-
methyl - A? - 1,3,4 - thiadiazoline - 2 - sul-
fonamide (CL 13,912), a new carbonic anhy-
drase inhibitor. Gzrorce M. S1sson* AND
Tuomas H. Maren. Exptl. Therapeutics Section,
Research Div., American Cyanamid Co., Stamford,
Conn.

In view of the interest in Diamox Acetazole-
amide for treatment of glaucoma and epilepsy, a
search was made for carbonic anhydrase inhibitors
with better penetration into the eye and brain.
Elimination of the dissociable hydrogen from the
carboxamide group of Acetazoleamide led to 1
such compound, CL 13,912. This substance is also
slightly more active than Acetazoleamide in vitro
as a carbonic anhydrase inhibitor. This compound
had greater activity than Acetazoleamide in ex-
perimental electroshock in the mouse (W. D.
Gray et al. Federation Proc., this issue) and in
lowering intraocular pressure in the rabbit (B.
BEcKER, personal communication). A single intra-
venous or oral dose of CL 13,912 at 5 mg/kg in the
dog gives a plasma concentration of drug above
2y/ml for 6-12 hr. Such concentration is associated
with renal effects typical of carbonic anhydrase
inhibition. Twenty to 40% of administered drug
is excreted in the urine unchanged. Renal clear-
ance of CL 13,912 is about 7 ml/min. or } that of
Acetazoleamide. CSF/plasma and aqueous/plasma
humor ratios of drug are about 0.3; the correspond-
ing figure for Acetazoleamide is 0.05. In other
species also CL 13,912 is superior to Acetazole-
amide in penetration into the brain (cat, mouse)
and CSF (cat, man). The pharmacology of CL
13,912 in man is similar to that described for the
dog. Long-term studies in the dog show that at
33 mg/kg in single daily oral doses CL 13,912 is
well tolerated for at least 7 months.

e 16

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March 1966

1577. Fate of papaverine. ALBERT SJOERDSMA,*
Jutius AxELRoD, RoBert SHoFEeR,* WILLIAM
M. Kina* anv Joun D. Davipson.* Natl. Heart
Inst. and Natl. Inst. of Mental Health, Bethesda,
Md.

A specific and sensitive method for the estima-
tion of papaverine in biologic material has been
developed. The physiological disposition of the
drug was studied in man and animals. In man,
papaverine was rapidly and completely absorbed
from the gastrointestinal tract and only a trace of
the drug was excreted unchanged in the urine.
After the intravenous administration of 3 mg/kg,
the biological half life in plasma was found to
range from 1 to 2 hr. in 3 human subjects. A rela-
tively constant plasma level was maintained by
oral administration of 200 mg papaverine every
6 hr. for 6 days. Tissue distribution studies in dogs
showed a considerable localization of the drug in
fat depots and liver with uniform distribution in
other tissues including brain. At therapeutic
plasma levels the drug was found to be bound to
plasma proteins about 90%. Comparative studies
on the physiological disposition of papaverine and
paveril have also been done in man. Preliminary
results have indicated that papaverine is cleaved
by a microsomal enzyme system in liver to yield
formaldehyde and presumably a phenolic metab-
olite.

1578. Metabolism of pyrimethamine. Cart C.
SmitH, L. H. Scumipt, R. FRaDKIN* AND J.
Inria.* The Christ Hosp. Inst. of Med. Research,
Cincinnati, Ohio.

Previous studies in this laboratory have shown
that pyrimethamine is able to protect rhesus
monkeys against infection with the trophozoites
of P. cynomolgi when administered in single doses
of 1 mg/kg 4-8 days prior to inoculation. Since the
parent drug is rather rapidly eliminated by the
monkey via urinary excretion and/or degradation,
it has been postulated that the protracted protec-
tion of single small doses rests on conversion of
pyrimethamine to a metabolite which is retained
for considerable periods. Work outside this labora-
tory (Goopwin, Trans. Roy. Soc. Trop. Med. &
Hyg. 46: 485, 1952) suggests that such long-acting
metabolites may have greater ‘antifolic’ activity
than the parent drug. The present study was de-
signed to test this possibility through systematic
measurements of the pyrimethamine content and
‘antifolic’ activity of the urine of monkeys treated
with this drug. These studies showed the urinary
elimi:ation of substances with the capacity to
block the utilization of folic acid by Streptococcus
fecalis closely paralleled the elimination of
‘pyrimethamine’ as determined chemically. In no
instance was the ‘antifolic’ activity of urine
greater than would be anticipated from the

; XUM

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

485

amount of ‘pyrimethamine’ present. This finding
suggests that the protracted protection from
single doses of the compound does not reside in
the conversion of the drug to a long-acting folic
acid antagonist more potent than pyrimethamine
itself. This suggestion finds support in the obser-
vation that neither folic nor folinic acid blocks the
activity of this drug against cynomolgi malaria in
the monkey.

1579. Effects of acetylcholine and atropine on
stretch receptors in frog muscle. CEDRIC
M. SmitH AND Kuaus R. Unna. Dept. of Phar-
macology, Univ. of Illinois College of Medicine,
Chicago.

The effects of acetylcholine and atropine sulfate
on stretch receptors were assessed on the isolated
peroneal nerve-extensor longus digiti IV muscle
preparation. The entire muscle and nerve were iso-
lated and placed in a 50-ml bath; the muscle was
stretched with a 1 gm load applied to the tendon;
tension was recorded isometrically via a strain
gage pick-up, carrier wave amplifier and recorder.
Nerve action potentials were recorded oscillo-
graphically from silver-silver chloride electrodes
placed on the peroneal nerve. The addition of
acetylcholine (0.7-40.0 ug/ml) caused a marked
increase in the rate of stretch receptor discharges
as recorded from the afferent nerves; this effect
was maximal 2-10 sec. after the addition of acetyl-
choline. The frequency of discharges returned to
the control rate within 1 min. Atropine (2.0-40.0
ug/ml) completely abolished the effects of acetyl-
choline. Dose-response and time-effect curves will
be presented. Acetylcholine, in concentrations
greater than those required to increase the rate of
stretch receptor discharges maximally were
capable of producing a contracture of the muscle;
however the maximum tension observed with a
contracture was less than 75 of the tension of a
muscle twitch elicited by single square wave
stimuli applied to the nerve. Atropine, in concen-
trations which blocked the action of acetylcholine
on the stretch receptors, had no observable effects
on muscle twitches induced by nerve stimulation.
(Supported in part by Grant B-973 from the
Public Health Service.)

1580. Interrelations between potassium, his-
tamine and acetylcholine upon the reac-
tivity of isolated arteries. DuRwoop J. SMITH
AND WiLui1AM H. Macmi.Luan.* Dept. of Phar-
macology, Univ. of Vermont College of Medicine,
Burlington.

It has been shown that injections of histamine
into animals results in an explosive release of
potassium into the serum. These observations
made it necessary to define the interrelationships
between histamine, potassium and acetylcholine

486

upon reactivity of the peripheral vascular system.
Variations in arterial reactivity caused by these
agents have been studied in isolated surviving dog
and swine arteries employing an angioplethysmo-
kymorgraphic technique (Circulation 4: 890, 1951).
Perfusion of vessels with histamine (1 ppm) in-
creased the vascoconstriction produced by acetyl-
choline in the vessels studied. An increase in the
K* ion in the perfusate from 3.0 mE/I to 4.0 mE/1
increased the sensitivity of isolated vessels to
histamine and acetylcholine 50%. A decrease in
K* ion decreased vascoconstriction below control
responses. These in vitro results will be discussed
in terms of the in vivo cardiovascular response to
ionizing radiation. (Supported by USAFSAM
Contract #*AF 19(604)-1093 and PHS Grants
B 729 and H-1450 C).

1581. Enzymatic transformation of digitoxin
studied polarographically. BarsBara A.
Sotomon* anp F. H. Meyers. Dept. of Phar-
macology and Exptl. Therapeutics, Univ. of Cali-
fornia Med. Ctr., San Francisco.

The amount of digitoxin in a 50% alcohol solu-
tion can be determined polarographically using
0.1 m tetraethylammonium hydroxide as support-
ing electrolyte and using a mercury pool as anode,
following the method of Fieser and Hilton. Digi-
toxin is added to rabbit liver prepared by grinding
with sand and extracting with buffer, and after
incubation the chloroform extractable material is
polarographed. The wave characteristic of digi-
toxin (E 4 = —1.85 to —1.95 V) is rapidly trans-
formed and a new wave (E 4 = —1.45 to —1.55)
appears. Since digitoxigenin and isodigitoxin have
half waves indistinguishable from that of digi-
toxin, the transformation is not hydrolysis of the
glycoside bonds or conversion to isodigitoxin. In
strong aqueous base, which is known to open lac-
tone rings, the behavior of digitoxin is similar to
that produced by liver but the reaction is much
slower. Schwear’s work, suggesting that the half
wave characteristic of digitoxin depends upon the
carbonyl group accompanied by an adjacent un-
saturation, also indicates that the change must
involve tlie lactone ring. (Supported, in part, by a
grant from the Natl. Heart Inst.)

1582. Metabolism of thiouracil by the rat.
EvuiotT Spector* ANpD F. E. SuHrpeman. Dept. of
Pharmacology and Toxicology, Univ. of Wiscon-
sin, Madison.

The existence of an enzymatic mechanism with-
in the liver of the rat, capable of converting cer-
tain substituted thiobarbituric acids to their
oxygen analogs, has been demonstrated previ-
ously (WinteERS ef al. J. Pharmacol. & Exper.
Therap. 114: 343, 1955 and Specror AND SHIDEMAN,
J. Pharmacol. & Exper. Therap. In press). The
following experiments were performed to deter-

FEDERATION PROCEEDINGS

Volume 16

mine whether or not thiopyrimidines undergo a
similar metabolic transformation. The simplest
member of this group, thiouracil (100 mg/kg), was
administered intraperitoneally to female albino
rats of the Holtzman strain. The urine from these
animals, after extraction with petroleum ether,
then chloroform, was examined by ion exchange
and paper chromatography and revealed the
presence of significant amounts of uracil. When
thiouracil (1 mg) was incubated aerobically in a
buffered medium with a mince or homogenate of
rat liver (1 gm) at 38°C for 3 hr., 28-35% of the
drug was metabolized. During this period of time
significant amounts of ammonia, representing ap-
proximately 10% of the metabolized drug, were
formed. After acidification of the incubated mix-
ture, the supernatant fluid was concentrated by
evaporation and subjected to paper chromato-
graphic analysis in 2 different systems of phenol
and water. When the paper was sprayed with a
solution of copper sulfate, dried and then sprayed
with a solution of diphenylthiocarbazone, the
presence of a material with the same Rr values as
those for B-alanine was revealed. By employing
appropriate ion exchange and paper chromato-
graphic technics, the aqueous phase of the incu-
bated mixture also was shown to contain uracil in
significant quantities. These findings suggest a
metabolic pathway in the rat for thiouracil which
involves first its desulfuration with the formation
of uracil followed by cleavage of the pyrimidine
ring and formation of 8-alanine and ammonia.

1583. Effect of various mitotic inhibitors on
oxidative phosphorylation. SypNEyY Spector*
AND KwanaG Soo Lee. Dept. of Pharmacology,
Jefferson Med. College, Philadelphia, Pa.

The effects of various mitotic inhibitors were
studied on oxidative phosphorylation in rat liver
mitochondrial preparations made in_ isotonic
sucrose. At a final concentration of 0.2 m urethane
decreased the P:O ratios when a-ketoglutaric acid
was employed as a substrate under aerobic condi-
tions. Colchicine under the same experimental
conditions exerted very little effect. Nitrogen
mustard (methyl bis (B-chloroethyl) amine HCl)
did not have any effect on oxidative phosphoryla-
tion until a concentration of 5 X 10-3 m was
reached. Above this concentration the depression
of oxidation was such as to suggest a nonspecific
uncoupling by the drug. The individual steps in
the electron transport system were then analyzed
to localize the sensitive site. Urethane at a con-
centration of 0.2 m produced a decrease of the
P:O ratio when succinate was utilized as a sub-
strate aerobically, as well as anaerobically when
ferricyanide was used as the artificial electron
acceptor. Colchicine at a concentration of 10? M
appeared to inhibit the phosphorylation coupled
to the oxidation of ascorbic acid. The effective con-

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March 1956

centrations of the drugs on oxygen uptake of
Ehrlich Ascites Tumor cells and of the mito-
chondrial preparations were nearly identical.
(Supported in part by a grant from the Natl. Inst.
of Health.)

1584. Changes in radioactivity of proteins and
fats following exposure to drugs. FREDERICK
Speruina. Natl. Inst. of Arthritis and Metabolic
Diseases, Natl. Insts. of Health, Bethesda, Md.
Developing frog ova and embryos whose pro-

teins and fats were tagged with C™ prior to ovula-

tion (SPERLING, Anat. Rec. Suppl. 120: 1954;

Nature 174: 749, 1954), were exposed to dinitro-

phenol, phenobarbital and pentobarbital. After

exposure to these drugs for varying periods of
development, the proteins and fats were extracted
and the radioactivity measured and compared to
that of nonexposed specimens. In the unexposed
specimens, during development from cleavage to

Shumway stage 18, at which muscular response

begins, the radioactivity of the proteins remained

relatively stable, while that of the fats followed a

logarithmic decay curve. After exposure to di-

nitrophenol, the radioactivity of the proteins

dropped sharply, while that of the fats dropped
only slightly, but still followed a decay curve
similar to that of the unexposed specimens. Bar-
biturate exposure, on the other hand, was followed
by higher activity in both proteins and fats than
that found in unexposed specimens. These changes
in radioactivity were not associated with changes
in the weight of proteins and fats. The stage of
development of the ova at the time of exposure
was a factor in the degree of response to the drug.

The greatest response occurred when the ova were

exposed during gastrulation.

1585. Oxidative phosphorylation in liver mito-
chondria of hypothyroid rats. M. A. SprrTes
AND A. ANDOosE,* Div. of Pharmacology, Hahne-
mann Med. College, Philadelphia, Pa.

The physiological importance of the in vitro
effects of thyroxine on mitochondrial prepara-
tions has been questioned by a number of workers.
For example lowering of the P:O ratio can be pro-
duced equally as well by L- or piL-thyroxine ac-
cording to Bain (J. Pharmacol. & Exper. Therap.
110: 2, 1954). Furthermore, in most cases, the
thyroxine added does not increase oxygen con-
sumption. The level of thyroxine needed (10 m)
is much higher than the amounts normally present
in body tissues. Finally, the thyroxine effect can
be overcome by high Mg*+ concentrations. It was
therefore of interest to determine whether oxida-
tive phosphorylation in hypothyroid rats was
higher than in normal controls. Wistar rats weigh-
ing 100-150 gm were made hypothyroid by thyroid-
ectomy followed by the injection of 250 ue of I!
intraperitoneally or by injecting 750 ue of I! with-

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

487

out operation. Jn vivo metabolism was followed by
CO: exhalation studies. Metabolism dropped
15-25% after 3-4 wk. The animals were killed by
cervical dislocation and liver mitochondrial prep-
arations made by the sucrose procedure. A mito-
chondrial oxidative system with a-ketoglutarate
as substrate according to Kielley (J. Biol. Chem.
191: 487, 1951) was used. Jn vitro oxygen consump-
tion in such preparations using no ATP acceptor
system fell 20-40% when compared with controls.
In the same mixtures, using malonate and fluoride
inhibitors, plus an ATP acceptor system, the P:O
ratio was 3.5 for 9 hypothyroid animals as com-
pared to 2.5 for 9 normal controls. (Supported by
Public Health Service Grant A-580).

1586. Prolonged gluco-corticoid activity meas-
ured by thymus gland regeneration and
protection against lethal anaphylaxis in
mice. M. T. SPOERLEIN AND S. MARGOLIN (in-
troduced by Go Lu). Pharmacological Research
Dept., Schering Corp., Bloomfield, N. J.

In thymus regeneration experiments, normal
male Carworth mice (18-24 gm) received single
graded doses of steroids subcutaneously. At inter-
vals, thereafter, wet thymus weights were deter-
mined for 8 mice at each steroid level, and for
controls. With maximal thymus involution present
by the 4th day, a regeneration curve was derived
from the successive mean thymus weights. At 30
mg/kg, thymus weights were again normal after
13 days with cortisone acetate, and after 19 days
with cortisone diethylacetate. At 120 mg/kg,
thymus weights returned to control values by the
19th day with cortisone acetate, but continued at
maximal involution weights after 19 days with the
diethylacetate. The 2 esters did not differ in
potency on the 4th day. However, by the 8th,
diethylacetate was 1.6 times as active, and by the
13th, 4.4 times. In anaphylaxis experiments, ma-
ture male Carworth mice were sensitized as de-
scribed by Spoerlein and Margolin (Federation
Proc. 13: 408, 1954). Eighteen, 36 and 48 hr. after
the steroid injection, 5-10 sensitized mice/dose
were challenged with horse serum. The 2 intra-
muscular steroid dosages were 0.54 and 0.18
mg/mouse. For the respective doses of cortisone
acetate, protection was 100% and 80% at 18 hr.;
47% and 27% at 36 hr.; 30% and 0% at 48 hr.
Cortisone diethylacetate protected 100% and 80%,
respectively, at 18 hr.; 80% and 80% at 36 hr.; 80%
and 40% at 48 hr. Thus, the diethylacetate ester
of cortisone is significantly longer-acting than
acetate in both the thymus regeneration and
anaphylaxis experiments with mice.

1587. Thioctic acid in the treatment of hepatic
coma. FREDERICK STEIGMANN AND SHIBLI M.
Cananuati.* Hektoen Inst. for Med. Research of
the Cook County Hosp., Chicago, Ill.

488

The treatment of hepatic coma has remained an
enigma. Various new substances have been pro-
posed in addition to the usual liver regimen but
the mortality due to hepatic coma has remained
almost unchanged. The reason for this is that the
exact basis of hepatic coma is still unknown or
rather that hepatic coma probably is a syndrome
due to a combination of variable factors. Never-
theless, some of the new agents recently intro-
duced seem to have had some effect in certain
cases of hepatic coma. Recently thioctic acid has
been reported by some German workers as being
efficacious in patients with hepatic failure. Ac-
cordingly, we have used this material to date, in
11 cases of hepatic coma developing in patients
with cirrhosis (10) and in 1 patient with lepato-
spiral infection. We have used 2 cc of thioctic acid
intravenously as an initial dose daily and then
twice daily until the patient returned to con-
sciousness. Of the 11 patients, 6 died in hepatic
coma without response to the drug and 5 re-
covered. One of the cases recovered from coma
and died 5 days later without any determinable
cause. A 2nd patient died several days later of a
massive hemorrhage. Of the remaining 3 cases,
2 have left the hospital while 1 is convalescing as
yet in the hospital. From our experiences with
hepatic coma this ratio of recoveries seems to be
the most favorable one encountered. This sub-
stance should, therefore, be given a wider trial in
the treatment of hepatic coma.

1588. A new senna preparation in the treat-
ment of chronic constipation. FREDERICK
STEIGMANN. Gastro-Intestinal Clinic of the Cook
County Hosp., Chicago, Ill.

Despite the long use of Senna medically and as

a domestic remedy, its active principles were un-

known until Straub (1936) and Stoll (1941) re-

ported the isolation of sennoside A and B. Pharma-
cological studies indicated that the senna glyco-
sides pass-through the stomach unchanged, are
absorbed in the small intestine and slowly excreted
in the colon where by bacterial action a.substance
is produced that stimulates peristalses. through

Auerbach’s plexus. Objections to the use of senna

were griping, occasional nausea and wide varia-

tions in potency in different preparations due to
the impossibility of preparing a fully active and
stable liquid sennoside extract. The latter has

recently been overcome with the preparation of a

stable dry powder from the pericarp which con-

tains not only sennosides A and B, but also a third
sennoside, C described by Fairbairn, and the re-
maining as yet unidentified factors which add to
the total senna effect. Clinical trials by English
workers with the above preparation—Senokot—
gave good results and few side effects. Studies on

65 constipated patients showed in 62 good effect

with 1} to 1 teaspoon nightly; in 1 no effect and in

FEDERATION PROCEEDINGS

Volume 16

2 effect only after 3 teaspoons. The latter 2 had
occasional cramps. Preliminary motility studies
indicate a more rapid colon emptying after
Senokot. Our observations are similar to those in
England and suggest that Senokot might prove
itself useful in the regimen of chronic constipation,

1589. Isotope assay of intrinsic factor activity
of human gastric juice after its fractiona-
tion by continuous paper-electrophoresis,
Louxia STEPHANSON, Marityn Ricu, Roger
LAUGHTON AND GrEorGE B. Jerzy Guass (intro-
duced by Linn J. Boyp). Gastroenterology Re-
search Lab., New York Med. College, New York
City.

Gastric juices were aspirated after stimulation
by histamine or insulin from 9 normal subjects
and patients with duodenal ulcer, dialyzed under
refrigeration and lyophilized. Thirty to 100 mg of
dry material derived from each of the juices was
applied to the center of the paper curtain of
Durrum-type apparatus for continuous electro-
phoresis, and run in acetate (pH 4.0) or borate
(pu 9.2) buffer for 24 hr. All fractions collected
into each of the 30 tubes during each of the 25
fractionation experiments were analyzed for pro-
tein content by tyrosine method, and for nonhex-
osamine polysaccharides by the Badin modifica-
tion of the Shetlar method. Usually 3-4 fairly co-
incident protein and carbohydrate peaks were
obtained. The peaks were spread on both sides of
application point. The contents of all tubes
corresponding to each of the peaks in each of the
fractionation experiments were pooled, and their
intrinsic factor activity was assayed on patients
with pernicious anemia in remission by scintilla-
tion measurement of the hepatic uptake of CoB,
(Arch. Biochem. 51: 251, 1954). In each of the
gastric juices the intrinsic factor activity was
present in 2 or more fractions, usually located on
both sides of the application point and frequently
containing as little as 2 mg of dry material. In
view of such distribution of intrinsic factor ac-
tivity on continuous electrophoresis it appears
that the ‘pure intrinsic factor’ must exhibit ac-
tivity at a dose much below that of 2 mg obtainable
at the present. Continuous electrophoresis was
unable in our hands to fractionate gastric juice
into pure components. Intrinsic factor apparently
travels together with other mucoproteins of
gastric juice in the electrical field, unless the
intrinsic factor represents only a radical, chain,
or prosthetic group of mucoproteins which are en-
dowed with intrinsic facror activity. (Supported
by Grant-in-Aid A-68 (C3) from the Natl. Inst. of
Arthritis and Metabolic Diseases, PHS.)

1590. Antagonism of nicotine-induced convul-
sions by ganglionic blocking drugs. C.E£-
MENT A. SToNE, KATHERINE L. MECKLENBURG*

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March 1956

AND Mary L. Torcurana.* Pharmacology
Section, Sharp & Dohme, Div. of Merck & Co.,
Inc., West Point, Pa.

The known antinicotinic convulsive effects of
ganglionic blocking drugs have been studied
further in an effort to gain more information re-
lating to the site and mechanism of action. The
relative order of potency in inhibiting the con-
vulsant and lethal effects of nicotine in mice was:
3-methylaminoisocamphane (mecamylamine) >
pentolintum > hexamethonium. 4,5,6,7-Tetra-
chloro-2-(2-dimethylaminoethyl)-indoline (chlo-
risondamine) was incapable of antagonizing the
clonic convulsive events of nicotine, but was as
active as pentolinium in inhibiting extensor con-
vulsions and mortality. Tetraethylammonium was
inactive against any phase of nicotine convulsive
phenomena. On the other hand, chlorisondamine
was the most active peripheral ganglionic blocking
agent, as judged by its ability to dilate the pupils
of mice. It was followed by pentolinium, mecamyl-
amine and hexamethonium, in that order. Tetra-
ethylammonium was again inactive. The lack of
parallelism in order of potency between these 2
activities of ganglionic blocking agents suggests
that the anticonvulsant action was not related to
an indirect effect resulting from peripheral
ganglionic blockade. Curare-like effects also ap-
peared not to account for the anticonvulsant
effects, since none of the agents antagonized
strychnine-, Metrazol- or electroshock-induced
convulsions in mice. The tentative conclusion was
reached that ganglionic blocking agents antag-
onize the convulsant effects of nicotine at a CNS
site of action. This was supported by the fact that
the most potent antagonist was a nonquaternary
derivative of ammonium, the diffusion of which
into the central nervous system might be antici-
pated to be greater than for quaternary ammo-
nium blocking agents.

1591. Chromatographic separation of excre-
tory products after meralluride adminis-
tration in normal subjects. J. R. Strawn.*
C. A. Hanpiey, B. Kent* anp R. A. SEIBERT.
Dept. of Pharmacology, and Medicine, Baylor
Univ. College of Medicine, Houston, Texas.
Organomercurial compounds are believed by

some to produce their action by releasing mercuric

ions. The inorganic mercury is presumed to act on
the kidney tubules to block sodium chloride re-
absorption. The difference in potency of various
organomercurial compounds tends to disprove
this idea. We have undertaken to analyze urinary

excretion of meralluride when administered i.v.,

im. and s.c. by the following method: urine was

collected for control periods then at 6-hr. intervals

for 24 hr. after the administration of 2 cc (78 mg

Hg) of meralluride. Since meralluride is a sodium

salt of a carboxylic acid, it seemed possible that

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

489

an acid adsorbent would selectively retain the
compound and that degradation products and in-
organic mercury would pass through the column.
Acid aluminum oxide, an anionic adsorbent, was
selected. With this adsorbent known amounts of
meralluride and inorganic mercury could be
quantitatively separated. After administration
of meralluride, urine was passed through the
chromatography column followed by distilled
water to quantitatively remove the altered form.
Sodium carbonate followed by distilled water was
used to elute the adsorbed fraction. These two
fractions as well as the untreated urine were then
analyzed for mercury using the method of Lang
and Nelson (J. Am. Official Agr. Chem., 25: 399,
1942). Animal experiments done by Seibert and
Handley revealed recovery of 80-100% of the in-
jected mercury in 6 hr. and of this, only 1-5%
passed through the column. With human subjects
using various routes of administration, results
were very similar. Up to 50% of the injected
mercury was recovered in 6 hr. In the next 18 hr.
up to 100% of the injected mercury was recovered.
Of this, only 2-5% was an altered form which
passed through the chromatography column. The
nature of this form or forms is yet unknown. It
leads us to believe that the action of organomer-
curial compounds is dependent on their organic
nature and not on the release of ionic mercury as
this amount is too small to be responsible for the
diuresis.

1592. Influence of ataraxics on the mescaline-
response of cats injected in lateral ventricle
of the brain. F. M. Sturtevant* anp VIcTOR
A. Dri. Div. of Biological Research, G. D. Searle
& Co., Chicago, Til.

Intraventricular injections of drugs in cats were
made after the method of Feldberg and Sherwood
(J. Physiol. 123: 148, 1954). Mescaline alone (1-3
mg) caused an immediate succession of yowling,
tachypnea, emesis, defecation and mydriasis,
followed by a prolonged period of catatonic stupor.
Reserpine (0.1 mg) caused a mild ataraxia (tran-
quility) unaccompanied by miosis or relaxation of
the nictitating membrane; 2 hr. later, mescaline
produced its immediate effects, but not catatonia;
the following day the cats had a typical ‘reserpine-
hangover’: miosis, nictitating membrane relaxa-
tion, tachypnea, anorexia. Chlorpromazine (2 mg)
produced nictitating membrane relaxation,
tachypnea, ataxia and catalepsy; cats that re-
ceived only 1 mg and were completely normal
several hours later responded to mescaline only
with a moderate decrease in spontaneous activity.
Azacyclonol (1-5 mg) caused tachypnea, emesis
and moderate ataraxia; several hours later, mes-
caline produced its typical immediate effects fol-
lowed by excitatory behavior in 4 out of 5 cats.
It was concluded that there ataraxics showed

490

anti-mescaline activity in the following decreas-
ing order of potency: chlorpromazine, reserpine
and azacyclonol.

1593. Cardiovascular actions of choline chlo-
ride. Henry H. Swain (introduced by T. M.
Bropy). Dept. of Pharmacology, Univ. of Michi-
gan, Ann Arbor.

The cardiovascular actions of choline chloride
have been reinvestigated. In the dog anesthetized
with pentobarbital, intravenous doses of choline
of 15 mg or less produce a transient, marked fall
in blood pressure. With larger doses of the com-
pound, this depressor response is followed im-
mediately by a pronounced rise in pressure. This
pressor response is greater with choline than with
its common congeners. The depressor component
of the response is blocked by atropine, while the
pressor action is abolished by a combination of
acute adrenalectomy and hexamethonium. The
pressure rise after choline is still present after
adrenalectomy alone or hexamethonium alone.
After atropinization to prevent excessive mus-
carinic effects, successive doses of choline give
progressively smaller pressure rises until the
pressor response is blocked by an amount of
choline which also produces temporary respira-
tory arrest. Intraarterial injection of small doses
of choline produce a transient increase in femoral
artery blood flow, and this response is blocked by
atropine. In the dog heart-lung preparation, large
doses of choline alter both muscular contraction
and impulse conduction. The compound exerts a
positive inotropic effect, but to demonstrate this,
the muscarinic action upon A-V conduction must
be circumvented by atropinization or by elec-
trically driving the ventricle at a constant rate.
Normal intraventricular conduction is somewhat
prolonged. If conduction has been prolonged by
administration of poiassium salts, choline has the
opposite effect, exerting a marked positive dromo-
tropic action to return intraventricular conduc-
tion to normal values. (Supported by a grant from
the Michigan Heart Assoc.)

1594. Effects of two amino acids and their
amides on convulsions induced experimen-
tally. Irvine I. A. Taspacunick,* R. E. Par-
KER,* J. WAGNER* AND F. H. Scuuttz, Jr.
Baxter Labs., Inc., Morton Grove, Til.
Contradictory reports concerning the efficacy

of glutamic and aspartic acids or their amides in

experimental as well as human convulsions war-
ranted investigation of these substances. Gum
acacia suspensions or neutral solutions of the test
substances were administered intraperitoneally to
albino mice. The mice were then tested according
to the procedures of Swinyard et al. (J. Pharmacol.

& Exper. Therap. 106: 319, 1952). The substances

evaluated have little, if any, ability to protect

FEDERATION PROCEEDINGS

Volume 15

against maximal electroshock convulsions (MES)
or convulsions induced by subcutaneously ad-
ministered metrazol. In the MES test, 3200 mg/kg
of p- or L-glutamic acid or L-asparagine was re-
quired before any significant protection was ob-
served. Concomitant with this protection, general
toxicity approaching a comatose state was ob-
served. Other amino acids and amides tested were
ineffective. In the metrazol test, DL-aspartic acid
and L-aspartic acid were totally ineffective in
doses ranging up to 3200 mg/kg; 2000 mg/kg of
L-glutamine protected 20 per cent of the animals.
p- or L-glutamic acid, or L-asparagine, 1500 to
3000 mg/kg, afforded a maximum of 50% protec-
tion; higher doses could not increase the propor-
tion of animals protected. Only one substance,
D-asparagine, was capable of protecting more than
50% of the animals challenged with metrazol; at
2000 mg/kg, 80% of the mice were protected. The
ED,» for D-asparagine was 1360 mg/kg (probability
limits, 1046-1768). These studies suggest that the
substances tested have little value in protecting
mice against convulsions induced experimentally.

1595. Contracted kidney and hypertension in
rabbits following the injection of spermine
into the renal artery. CeL1A WHITE TaBor,*
LLEWELLYN L. ASHBURN* AND SaNFoRD M,
RosentTHAL. Natl. Insts. of Health, Bethesda,
Md.

Spermine, a naturally occurring polyamine, has

a marked acute renal toxicity, producing tubular

degeneration and death when administered

parenterally (Proc. Soc. Exper. Biol. & Med. 80:

432, 1952). It has now been demonstrated that a

single injection of spermine hydrochloride (75

uM/kg) into the left renal artery of rabbits pro-

duces progressive degeneration leading to marked
atrophy of the injected kidney and hypertrophy
of the opposite kidney; 8-15 days after the injec-
tion, 9 out of 11 rabbits showed marked tubular
necrosis of the injected kidney. After 1-3 months

10 out of 12 rabbits showed interstitial fibrosis and

marked atrophy of the injected kidney; 4 of these

10 rabbits showed cardiac enlargement. Blood

pressure measurements by the ear cup method

were followed in 5 rabbits; these blood pressures
showed a gradual increase, and at 2 months after
the injection the average blood pressure reading
was 30 mm higher than control values. Preliminary
experiments with spermidine demonstrate com-
parable effects. No significant pathology or hyper-
tension was observed 1-2 mo. after the following
injections into the renal artery: saline (5 rabbits);
putrescine hydrochloride 75-150 um/kg (3); prota-
mine sulfate 15 mg/kg (1); and neutralized as-
partic acid 113 um/kg (1). Contracted kidneys
have not been noted following subcutaneous ad-
ministration of spermine. In preliminary experi-
ments with 2 rabbits, subcutaneous injection of

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March 1956

75-100 uM/kg of spermine at 10- to 44-day intervals
for 2 and 5 months respectively, resulted in only
moderate bilateral tubular atrophy and slight
NPN retention.

1596. Influence of 9-alpha fluorohydrocorti-
sone on electrolyte distribution and con-
tractility of isolated cat papillary muscle.
Rauvpeu D. Tanz aNnp GLENN M. CLaRK (intro-
duced by Richarp W. WuitenHEaD). Depts. of
Pharmacology and Medicine, Univ. of Colorado
School of Medicine, Denver.

The isolated, contracting cat papillary muscle
preparation of Cattell and Gold offers several ad-
vantages in the study of intracellular metabolism.
The muscle can be dissected without damaging the
cells, and will contract for 72 hr. under intermit-
tent stimulation. In addition the small, thin prep-
aration allows rapid transfer of gases and metab-
olites into and out of the muscle cells. In these
experiments the papillary muscle was stimulated
while bathed in Krebs-Ringer bicarbonate solu-
tion, maintained at a constant temperature of
38°C. The strength of contraction was recorded
by means of a transducer tube, capable of trans-
forming mechanical displacements into electrical
potentials, which in turn were picked up by a
direct-writing Sanborn EKG. Either isotopic
sodium or potassium was used in the preparation
of the perfusing medium and the radioactivity of
the muscles determined at the conclusion of the
experiments. The uptake of isotopic Na or K was
expressed in counts/sec/gm and then in terms of
the ratio of radioactivity of experimental to con-
trol. The addition of 9-alpha fluorohydrocortisone
in concentrations less than 1.0 ug/ml. was fol-
lowed by a marked positive inotropic effect and
the simultaneous uptake of both Na and K ions.
In all instances the Na and K uptake ratio of ex-
perimentals to controls was greater than 1.0.
Thus, it has been shown that under the direct
effect of a steroid, other than glycosides or agly-
cones, a positive inotropic action was observed.

1597. Duration of anticonvulsant action of
trimethadione and some demethylated
oxazolidinediones against pentylenetet-
razol in mice. J. D. Taytor, JEANNE C.
Davin* AND R. K. Ricuarps. Dept. of Pharma-
cology, Abbott Labs., N. Chicago, Ill.

Doses of trimethadione that protect mice
against pentylenetetrazol convulsions are known
to have a long duration of action. Butler (J.
Pharmacol. & Exper. Therap. 108: 11, 1953) showed
that demethylated trimethadione (5,5-dimethyl-
2,4-oxazolidinedione) (DMO) was a metabolite of
trimethadione in the dog which persisted in the
body for relatively long periods of time. It was the
purpose of this work to ascertain if the long action
of trimethadione might possibly be due to DMO.

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

491

Groups of mice were given trimethadione and
DMO. At various time intervals up to 24 hr.),
100 mg/kg pentylenetetrazol was injected subcu-
taneously and both anticonvulsant protection
and deaths were noted. When the ET;» (median
effective time) for anticonvulsant protection was
plotted against dose 2 distinct lines with different
slopes were obtained with the result that at equal
doses the ratio of duration of anticonvulsant effect
of these 2 drugs changed with the dose. When
death was used as the criterion of response, the
dose-duration curves for these 2 drugs were not
significantly different in slope or position. Similar
experiments were carried out with paramethadione
and its demethylated metabolites.

1598. Response of various Neurospora strains
to azo dyes. P. 8. THayer,* D. C. MAHONEY*
AND C. J. Kenster. Arthur D. Little, Inc.,
Cambridge, Mass.

Salzberg (Proc. Am. A. Cancer Research 2(1):
43, 1955) reported that Neurospora mutant 51602,
which requires riboflavin for growth, is inhibited
by the carcinogenic azo dye 3’-methyl-4-dimethyl-
aminoazobenzene, and that this inhibition is pre-
vented by riboflavin. Adaptation to the dye occurs
so that (genetically) adapted cultures can be ob-
tained, the growth of which is stimulated or even
supported by the dye in the near or complete ab-
sence of added riboflavin. These results have been
confirmed, using 3/-methyl-4-methylaminoazo-
benzene (3’-Me-MMB). Further work in our labo-
ratory has shown that the inhibition is not limited
to the carcinogenic members (for the rat) of a
series of azo compounds, and that a significant
degree of inhibition of strain 51602 is produced by
the chemically dissimilar carcinogen, acetylamino-
fluorene. These effects are most pronounced at
temperatures above 28°C where the riboflavin
requirement of 51602 is absolute. The compounds
tested and the percent inhibition obtained with
each at comparable concentrations were: 3’-Me-
MMB, 57%; 2’-Me-MMB, 53%; 4’-Me-MMB, 47%;
MMB, 30%; AB, 30%, and acetylaminofluorene,
12%. Increased riboflavin prevented the inhibition
by all these to about an equal extent. Adapted
strains have been obtained by ‘training’, and
their responses to the series of inhibitory com-
pounds, and some of their breakdown products,
have been compared with those of 51602, other
mutants and a wild type, at different tempera-
tures, riboflavin levels and incubation periods.
Isolates from crosses of the adapted and unadap-
ted cultures of 51602 with wild type have also
been studied.

1599. Central inhibition in electroshock sei-
zure pattern, and its modification by stress
and drugs. J. E. P. Toman, G. M. Everett
AND A. H. Situ, Jr.* Dept. of Physiology,

492

Chicago Med. School, and Dept. of Pharmacology,

Abbott Labs., N. Chicago, Iil.

Correlations of seizure latency, duration and
recovery in 700 mice strengthen the concept that
electroshock initiates interacting excitatory and
inhibitory factors which determine seizure latency
and severity and persist into the recovery period.
They are differentially modifiable by various
stresses and drugs. Mechanical shaking at 6/sec.
for up to 80 sec. doubles latency, shortens or
abolishes tonic extension, shortens recovery
period and abolishes seizure fatalities. Results
mimic simple reduction of stimulus voltage, and
are not attributable to anoxia, hypercapnia, sym-
pathoadrenal discharge or central depression.
More prolonged shaking reverses these effects con-
comitant with central nervous impairment.
Reserpine or desmethoxyreserpine (10-100 ug/kg)
reduce latency markedly and duration somewhat,
but increase fatalities and recovery time. Shaken
reserpinized mice resemble reserpine controls in
short latency and high fatalities, but show in-
creased extensor phase and recovery time, plus
central depression with otherwise innocuous shak-
ing. Thus although reserpine (also desmethoxy-
reserpine and chlorpromazine) opposes stress
effects, it does so by tangential rather than direct
mechanisms and impairs central regulations.
Reserpine is thought to act predominantly on the
rate factor of inhibition. Phosphate somewhat
resembles reserpine in action, and hypercapnia
and hypercalcemia have partly opposite actions.
However, experience with these and other drugs
suggests a multiplicity of factors capable of modi-
fying seizure pattern. Qualitatively, all normal
components of seizure pattern are found in mice
decerebrated at a midcollicular level.

1600. Ethchlorvynol (Placidyl) compared with
secobarbital and pentobarbital for EEG
sedation. J. E. P. Toman anv. E. K. CHrIstTENn-
SEN,* Dept. of Physiology, Chicago Med. School,
and Mt. Sinai Hosp., Chicago, Ill.

Of 129 consecutive EEG service patients, 34 were
pretreated with ‘P’ (Placidyl, Ethchlorvynol), 62
with ‘S’ (Seconal, secobarbital), and 16 with ‘N’
(Nembutal, pentobarbital). Seventeen controls
received placebo or no medication. Usual doses in
mg for children and adults were: P 100, 250; S 50,
100; N 25, 50. If necessary, additional dosages were
given after 1 hr. (maximum total dose P 750, S 200,
N 200). Only 25% of controls remained awake
throughout the recording period (90-150 min.).
Sedatives approximately halved this number; S
was slightly more effective than others. Median
onset of sleep was 43 min. for all patients, with no
significant effect of medication. There were no
significant differences in EEG abnormalities, or
in sound-activated sleep potentials. All sedatives
significantly increased waking EEG fast frontal

FEDERATION PROCEEDINGS

Volume 16

activity, and also increased the number of patients
requiring forcible arousal. P produced signifi-
cantly easier arousal and less ‘grogginess’ than
barbiturates. Age was a significant factor; suc-
cessive quartiles (1-12, 13-30, 31-45, 46-77 years)
showed increasing sleep delay, easier arousal and
more failures. Despite special requirements, EEG
daytime sedation is considered advantageous for
quantitative drug evaluation.

1601. Effect of ganglionic blocking agents
upon mesenteric blood flow in the anes-
thetized dog. JoserpH H. TRAPOLD AND JOAN
G. Sutuivan.* Dept. of Pharmacology, Louisi-
ana State Univ., School of Medicine, New Orleans.
A reduction in mesenteric flow associated with

a concomitant increase in mesenteric resistance

has been reported to follow the intravenous ad-

ministration of the ganglionic blocking agent
chlorisondamine to anesthetized ‘normotensive’
dogs (PLuMMER, A. J. et al., 1955). A similar re-
sponse following the administration of pento-
linium and hexamethonium to dogs was reported
by Trapold (1955). In these earlier studies, blood
flow was measured using the technique of double
cannulation (inflow and outflow cannulae) of the
mesenteric artery and with the aid of a Shipley-
Wilson Rotameter. In an extension of these studies
in which the mesenteric artery was catheterized
via the left subclavian artery for outflow and the
left carotid artery employed for inflow, it was
found that although the above agents as well as
mecamylamine (0.5 mg/kg i.v.) produced a de-
crease in mesenteric flow, mesenteric resistance
either remained unchanged or decreased slightly.

With the latter method, the pressure in the out-

flow limb of the flow system was significantly lower

than the systemic pressure of the animal. With the
method of double cannulation of the mesenteri¢
artery, the difference in pressure between these

2 areas was much less pronounced. Since the dis-

crepancies between the results obtained by these

methods appears to be related to the degree of
pressure drop induced by the flow systems, this
factor is now under investigation. (Supported,

in part, by Public Health Service Grant H-2030

and Ciba Pharmaceutical Co.)

1602. Effect of certain narcotics on oxidative
phosphorylation. Epwarp B. Truitt, JR.
ARTHUR WOLPERT,* FREDERICK K. BELL* AND
JoHun C. Krantz, Jr. Dept. of Pharmacology,
Univ. of Maryland School of Medicine, Balti-
more, Md.

The uncoupling of oxidative phosphorylation
from respiration by a series of representative nar-
cotic agents was investigated using mitochondria
isolated from whole rat brain by a modification of
the method of Brody and Bain (J. Biol. Chem.
195: 685, 1952). Drugs were tested in concentra-

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March 1956

tions up te and slightly above accepted thera-
peutic levels. In a series of aliphatic alcohols from
methanol to pentanol uncoupling activity of a
significant degree occurred only with n-pentanol
and was slight (-—17% P:O ratio). Although
ethanol was inactive its metabolite, acetaldehyde,
showed significant activity. Other drugs tested
and found inactive were: paraldehyde, chloral
hydrate, tribromoethanol soiution, chlorobutanol,
methylparafynol, urethane, benzyl alcohol and
morphine sulfate. Uncoupling activity in the
system was confirmed by positive results with
pentobarbital sodium, Ca*+ and dinitrophenol.
(Supported in part by a grant from the Ohio
Chemical Div. of Air Reduction Co.)

1603. Uptake of reserpine-C' by various
areas of the cat brain. Wen Hur TsteEn,*
E. B. Stea,* Hersert SHEepparp,* A. J. PLum-
MER AND J. A. ScHNEIDER. Research Dept.,
Ciba Pharmaceutical Products Inc., Summit,
N.J.

Reserpine labeled with C in the 4-methoxy
carbon of the 3,4,5-trimethoxybenzoic acid
moiety, was injected into the carotid arteries of
each of six cats divided into 2 groups. One group
was killed at 10 min. and the other at 60 min.
after injection. The brains were excised as rapidly
as possible and sectioned into various functional
areas. No single area on a wet-weight basis
showed a concentration of C4 that differed signifi-
cantly from the others. The C™ concentration
tended to be somewhat higher at 10 min. than at
60 min. after injection. The tranquilizing effect
of reserpine, however, was not apparent until
approximately 50 min. after injection. Thus it
appears difficult to associate the concentration of
reserpine per se in the brain with its tranquilizing
effect.

1604. In vivo inactivity of some potent cho-
linesterase inhibitors. J. R. TurReEMAN,*
H. D. BatpripGe,* E. B. Coox,* 8. L. Frress*
AND D. J. JENDEN. Naval Med. Research Inst.,
Bethesda, Md.
1-(2-dimethylaminoethyl)-piperidine (I) and

Trimethyl (2-piperidinoethyl) ammonium iodide

(II) inhibit isolated electric eel cholinesterase

equally, and 4 times more strongly, than physo-

stigmine (III) respectively (Frress aND Mc-

CarviLLE, J. Am. Chem. Soc. 76: 1363, 1954). I,

II and III potentiated acetylcholine on the frog

rectus abdominis equally in concentrations of

10-* M, 1.2 X 10-3 M and 1.1 X 10-6 M, respec-

tively. I and II did not potentiate acetylcholine

on the rabbit ileum; III was effective at 3 X 10-®

M. I and II antagonized tubocurarine on the

guinea pig diaphragm at 4 X 10-4 M; III at 6 X

10-7 M. Similar inactivity was shown by I and II

-XUM

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

493

in intact animals; the intraperitoneal LD 50’s in
mice were 336+9 mg/kg, 407412 mg/kg and
0.860+0.070 mg/kg for I, II and ILI, respectively.
Signs of anticholinesterase poisoning preceded
death in I and III but not in II. The depressor
response to intravenous ,acetylcholine (0.27) in
anesthetized cats was transiently and minimally
prolonged by 4 mg/kg I or 2 mg/kg II. A greater
and longer-lasting effect was produced by 0.1
mg/kg III. The inactivity of I and II on living
tissues is not due to species specificity of cholines-
terase; both are } and 3 as active against serum
and erythrocyte cholinesterase respectively as
against electric eel acetylcholinesterase. The
discrepancy is too great to be explained in terms
of metabolism by any known enzyme in vivo. The
explanation appears to lie in non-specific binding
by a tissue and/or plasma constituent. In support
of this, 0.05% gelatin was found to protect pure
acetylcholinesterase against I by a factor of about
600.

1605. Increase in tissue serotonin by ad-
ministration of its precursor, 5-hydroxy-
tryptophan. SipNey UpENFRIEND, DoNaLp F.

BoGpDANSKI AND HERBERT WEISSBACH (in-
troduced by Brernarp B. Bropie). Lab. of
Chemical Pharmacology, Natl. Heart Inst.,
Bethesda, Md.

It has been previously shown that serotonin is
derived from tryptophan and that the inter-
mediate, 5-hydroxytryptophan (5HTP), is con-
verted to serotonin by a specific decarboxylase
found in many mammalian tissues. When C!*
labelled 5HTP was administered to rabbits the
serotonin in the body depots became so highly
labeled as to suggest an actual increase in the
total amount in the tissues. Administration of
non-isotopic 5HTP to animals, not only resulted
in an increase of serotonin in the usual depots,
but in the appearance of serotonin in tissues such
as liver, kidney, heart, and uterus, which nor-
mally contain no measurable amounts of this
substance. Chemical analyses indicate that 5HTP
is taken up by almost all body cells and there
converted to serotonin; 5HTP also penetrates
the blood brain barrier resulting in a marked
increase in brain serotonin. In animals it has
been possible to raise the level of brain serotonin
as much as twenty fold; the observed effects at
these high levels resemble those seen after ad-
ministration of the hallucinogenic drug, lysergic
acid diethylamide. The correlation of pharma-
cological effects with increases in brain serotonin
indicate that the activity of 5HTP results from
its decarboxylation to serotonin. This amino
acid precursor of serotonin should prove to be a
useful tool in elucidating the physiological role
of serotonin.

494

1606. Some factors involved in the response
of phosphatases of hematopoietic tissues
to x-irradiation. Epwin M. Uyeki* anp Pau.
R. Saterno. Atomic Energy Med. Research
Project, Western Reserve Univ., Cleveland,
Ohio.

The activity of several enzymes concerned with
the metabolism of adenosine nucleotides
(ATPase, 5-nucleotidase, adenylate kinase) in
hematopoietic tissues is markedly increased in
rats exposed to x-irradiation. Maximum change
is produced within 3 days post-irradiation and is
then followed by a gradual return to normal
activity within 3 wk. In view of a similar pattern
of response of these enzymes following irradia-
tion, the nature of this process of enzyme altera-
tion was further characterized. Cortisone acetate-
treated rats and starved rats showed a marked
rise in the ATPase activity of spleen and thymus
tissues. Adrenalectomized rats exhibited no in-
creased enzyme activity. The ATPase activity of
comparable numbers of cells obtained by forcing
splenic tissue through a fine wire mesh was found
to be increased more than 5-fold in irradiated
rats. To eliminate possible connective tissue
contamination, the effect of x-irradiation on the
ATPase activity of cells isolated from the blood
and lymph fluids was investigated. These studies
indicate that alterations in the ATPase activities
of leukocytes in irradiated rats reflect the changes
in the proportion of lymphocytes and neutrophils.
Changes in the proportion of neutrophils and
lymphocytes brought about by other means also
produced an alteration in the ATPase activity in
the peripheral blood.

1607. Analgesic tests based upon experi-
mentally induced acute abdominal pain in
rats. C. VANDER WENDE AND §. MARGOLIN
(introduced by Go Lu). Pharmacological Re-
search Dept., Schering Corp., Bloomfield, N. J.
Acute abdominal pain follows intraperitoneal

injection of aqueous solutions of sodium iodo-

methamate or the diethanolamine salt of iodo-
pyracet in rats. Other iodinated organic contrast
agents catse a similar reaction. The endpoint
indicating pain is a distinct, severe contraction
of the abdominal musculature accompanied by
writhing of the body, and backward extension of
hind limbs. Female rats (180-250 gm) received
the drugs orally 1 hr. (30 min. with subcutaneous
doses) prior to the intraperitoneal injection of

0.5 ml of a 25% solution of sodium iodometha-

mate. Three or 4 non-toxic graded levels of the

analgesics were employed to obtain a dose-re-
sponse curve. The oral EDso’s (mg/kg) for mor-

phine-like analgesics were: morphine sulfate, 30;

meperidine hydrochloride, 64; codeine phosphate,

42. Subcutaneously, the EDs5o’s (mg/kg) for

morphine sulfate and meperidine hydrochloride

FEDERATION PROCEEDINGS

Volume 1§

were 4.8 and 7.8, respectively. Antipyretic anal-
gesics given orally were ineffective at maximal
tolerated doses
900; aminopyrine, 400; phenylbutazone, 350. The
pain reaction to sodium iodomethamate is blocked

by 0.5 ml intraperitoneally of suitable concentra- §

tions of procaine hydrochloride, and indicates the
peripheral action of the pain-inducing agent. The
median effective concentration for procaine hy-
drochloride administered 5 min. prior to the
sodium iodomethamate is 2.1% with confidence
limits (P = 0.05) of 1.78%-2.48%. Preliminary
experiments suggest relative activity and dura-
tion of action for local anesthetics also can be
compared by this method.

1608. Effects of ouabain and veratridine on
potassium exchange in the isolated guinea-
pig heart. Roper? L. Vick* anp J. B. Kaun,
Jr. Dept. of Pharmacology, Univ. of Cincinnati
College of Medicine, Cincinnati, Ohio.

Isolated guinea-pig hearts were perfused with
Krebs’ Ringer-bicarbonate solution with added
glucose. Perfusion rate was recorded. Perfusates
were analyzed for potassium concentration with a
Baird flame photometer. Contractions were re-
corded isotonically on a smoked drum. To deter-
mine inotropic effects, hearts were made ‘hypo-
dynamic’ with solution in which one-half of the
calcium was replaced by sodium. After control
observations were made, drug was added to the
reservoir. Increase in contraction height above
the ‘hypodynamic’ baseline was taken as indica-
tion of positive inotropic effect. To determine
effects of the 2 drugs on potassium movements,
hearts perfused with normal solution were al-
ternately driven for 3 min. at 4/sec., and allowed
to beat for 3 min. at their intrinsic (slower) rate.
The higher rate produced potassium loss; resump-
tion of the slower rhythm permitted potassium
re-entry. Addition of ouabain in concentrations
not producing systolic arrest prevented potas-
sium re-entry when driving was stopped. Vera-
tridine, in doses which did not produce systoli¢
arrest, had little effect on potassium re-entry.
The effects of ouabain and veratridine on potas-
sium transfer in the heart are consistent with
their effects on potassium movements in incu-
bated cold-stored human erythrocytes (KaHN
AND AcHESON, J. Pharmacol. 115: 305, 1955).
(Supported by Public Health Service Grant
H-1506 (C).)

1609. Lysis of dog erythrocytes in mildly
alkaline isotonic media. W1LLIAM J. WaAD-
DELL (introduced by Tuomas C. BurtLzEp).
Dept. of Pharmacology, Univ. of North Carolina,
Chapel Hill.

Dog erythrocytes undergo hemolysis in any
isotonic medium with a value of pu higher than

(mg/kg): acetylsalicylic acid, §

go ~s =-3 ©) Oo 26 | hUrhlhUCU

ve;
the
of

16]

rel;
sul
chl
hos

lume 1§

ic anal-
naximal

c acid, §

50. The

blocked

icentra-
ates the
nt. The
ine hy-
to the
ifidence
iminary
1 dura-
can be

ine on
uinea-

Kaun,
ecinnati

od with
added
‘fusates
. with a
ere re-
» deter-
‘hypo-
of the
control
to the
, above
indica-
termine
ements,
ere al-
allowed
r) rate.
‘esump-
tassium
rations
potas-
. Vera-
systolic
»-entry.
| potas-
it with
n incu-
(KaHN

1955).

Grant

mildly
. Wap-
UTLER).
arolina,

in any
er than

March 1956

7.6. The higher the value of pu, the more rapidly
and the more completely hemolysis occurs; at
pu 7.4 about 2 hr. are required for noticeable
hemolysis. This phenomenon does not occur
with the erythrocytes of man, cat, mouse, guinea
pig, rabbit, sheep, horse or ox. The erythrocytes
of all species studied were seen to approach a
spherical shape as the pH was increased above
7.5 and to become crenated at pH values below
7.3. The transition to a spherical shape in alkaline
media is not due to increase in volume but rather
to a contraction of the surface membrane. It is
suggested that the dog erythrocyte differs from
that of other species in that its membrane can
withstand less tension. (Investigation carried
out during tenure of a PHS Research Fellowship
with partial support from grant B-384 from Natl.
Insts. of Health, PHS.)

1610. Excretion of C'+-labeled colchicine. E.
J. WALASZEK AND J. J. Kocsis (introduced by
E. M. K. Gerune). Dept. of Pharmacology,
Univ. of Chicago, Chicago, Til.

When sublethal doses of biosynthetically
labeled colchicine were administered to mice,
rats, guinea pigs and hamsters, it was found that
the urinary excretion of colchicine was lower in
the rat (8%) and the guinea pig (3%) than it was
in either the mouse (8%) or the hamster (9%),
the latter 2 species being relatively resistant to
colchicine. In the 24-hour period following the
administration of labeled colchicine a consider-
able proportion of the radioactivity was re-
covered as CQO, in the rat (82%) and the guinea
pig (28%) while lower levels were found in the
hamster (9%) and mouse (5%). When the total
radioactivity in the urine, feces and respiratory
CO. was determined for the 24-hour period fol-
lowing colchicine administration, it was found
that the rat excreted a much higher portion of
the injected dose (94%) than either the guinea
pig (54%), mouse (49%) or the hamster (44%).
Previously it had been shown that the presence
of a tumor can modify the disposition of ad-
ministered colchicine in mice and in patients with
cancer (Federation Proc. 13: 418, 1954). To in-
vestigate the role of other factors that can modify
the disposition of labeled colchicine the effects
of bile duct ligation and of x-irradiation in mice
were studied.

1611. Clinical analgesic assay of dihydrohy-
droxymorphinone. 8. L. WALLENSTEIN* AND
R. W. Houpe. Memorial Ctr., New York City.
A double blind, crossover evaluation of the

relative analgesic effectiveness of morphine

sulfate and dihydrohydroxymorphinone hydro-

chloride (Numorphan) was carried out in 26

hospitalized patients with pain due to cancer.

The study consisted of a series of sequential ex-

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

495

periments each of which contained 2 doses of the
standard medication (8 and 16 mg of morphine)
and 2 doses of the test drug (Numorphan). Con-
secutive dose levels of the test drug were selected
from an extension of the log scale of the standard
medication and were raised or lowered in a series
of sequential ‘quartets’ in an attempt to obtain a
maximum amount of data within the range of
approximate analgesic equipotence of the mor-
phine standard. Sterile saline was also included
to establish the placebo-effect baseline. Data was
collected on 4 drug quartets in which doses of
Numorphan ranged from 0.5-4 mg. A total of
158 i.m. doses of each drug were administered.
Pain relief scores were based on the patients’
hourly reports of changes in pain intensity for a
6-hr. period after drug administration. All drugs
in the quartets were superior to the saline con-
trols. A highly significant common slope for the
2 drugs was obtained and deviations from this
slope were not significant. Insignificant differ-
ences between test and standard drug effects
indicated that the doses used were in the range
of approximate equipotence. The best estimate
of relative potency equated 1.12 mg of Numor-
phan to 10 mg of morphine sulfate with a range of
0.90-1.65 mg at the 5% confidence level.

1612. Spasmolytic activity of atropine. JOHN
W. Warp,* Epwin H. Gray* anp Lioyp W.
Hazueton. Hazleton Labs., Falls Church, Va.
The spasmolytic effects of l-hyoscyamine hy-

drobromide and d-hyoscyamine sulfate were

compared to atropine sulfate, using segments of

isolated guinea pig ileum. The l-isomer had a

specific rotation of —25° representing 88% purity,

and a melting point of 149°C. Specific rotation: of
the d-isomer was +26° representing 89.5% purity,
and the melting point was 200°C. Atropine sulfate
was U.S.P. quality. Essentially, the method con-
sisted of determining the minimum quantity of
each isomer, employing atropine as a standard,
which would be required 1) to relax and 2) to
prevent a standardized submaximal contraction
of the ileum produced by acetylcholine chloride.

For evaluation of relative potency, atropine was

arbitrarily assigned the value of 1.0. Results of

these investigations indicate relative potency

values for relaxant activity of 2.0, 1.0, and 0.009

for I-, dl-, and d-hyoscyamine, respectively. In a

similar manner values for preventive activity

were 1.5, 1.0, and 0.017. Repetition of these
experiments gave values of 1.8, 1.0, and 0.013 for
relaxant activity, and 1.9, 1.9, and 0.007 for pre-
ventive activity. An average of these figures
demonstrates that /-hyoscyamine was 1.8 times

more effective than the racemic mixture and 150

times more effective than d-hyoscyamine. The

spasmolytic action of the d-isomer could be
explained in each instance by the amount of

496

l-isomer known to be present in the sample. It is
concluded that the levo-form of hyoscyamine is
the only active spasmolytic principle of atropine.

1613. Resting potential and action potential
of frog heart as influenced by calcium lack.
FREDERICK WARE AND A. L. BENNETT (intro-
duced by A. R. McIntyre). Univ. of Nebraska,
College of Medicine, Omaha.

Isolated active hearts of Rana pipiens perfused
with normal Clark-Ringer solution have a mean
resting potential of 84.5 mv (485 determinations
on 29 hearts). The least and greatest observed
R.P.s were 60 and 105 respectively. The standard
deviation was 7.1. With normal Clark-Ringer
solution the mean action potential was 102.5 (421
readings on 29 hearts), and the standard deviation
was 5.7. In a series of 16 experiments after per-
fusion with calcium-free solution, the R.P.
promptly fell by an average of 3-4 mv, and the
A.P. by 6-7 mv. Calcium lack produced a marked
change in the time course of repolarization and
‘humpbacked’ A.P.s resulted. Normally maximum
—dv/dt averaged 33.5 v/sec. whereas after per-
fusion with Ca** free solution the —dv/dt was
40.7 v/sec. The difference is statistically highly
significant. These results indicate the possibility
that in the absence of Cat*+ the normal ‘Nat
transport mechanism’ undergoes modification.

1614. Some effects of chlorpromazine on
phospholipid turnover in brain. ARTHUR W.
WasE, JENS CHRISTENSEN AND EDWARD POLLEY
(introduced by B. Catesnickx). Hahnemann
Med. College, Philadelphia, Pa.

Previous studies have indicated chlorpromazine
(CPZ) to accumulate in rat brain, particularly the
hypothalamus. The present studies were directed
toward the observation of changes induced in
phospholipid metabolism by the administration of
CPZ. Preliminary studies with mice indicated a
reduced rate of incorporation of P#2O,” into the
phospholipids of whole brain. The specific ac-
tivities of the phospholipids (epm P*2/mg Lipid-P)
of the CPZ (8 mg/100 gm) treated mice were 71,
52 and 49% of control values at 1, 2 and 4 hr. after
treatment. In another study, the specific activities
of the phospholipids of rat brain were determined
at 24 and 48 hr. after the administration of P#O,”
(50 ue/200 gm) and CPZ (5 and 10 mg/200 gm).
Results indicated a decreased turnover of phos-
pholipids in the cortex, cerebellum and thalamus.
An increased incorporation of P*? into the phos-
pholipids of the hypothalamus was observed. In
vitro studies with brain homogenates showed
reduced incorporation of radiophosphorus into
phospholipids to the extent of 84, 37, 31 and 27%
of control values as the concentration of CPZ
added to the medium was varied from 10-5, 10-4,
10-2 to 10-1 M. Studies are underway to determine

FEDERATION PROCEEDINGS

Volume ij

the turnover of individual phosphatides in thé
cellular and extra cellular lipid fractions 9
cerebral tissue. (Supported by Smith, Kline and
French Laboratories.)

1615. Effect of smoking on urinary output ¢
epinephrine and norepinephrine in man. J),
T. Warts anp A. D. Braaa.* Dept. of Phar.
macology, West Virginia Univ. Med. School;
Morgantown.

The urinary epinephrine-norepinephrine output
of 17 non-smoking medical students and of
students who normally smoked was determined
for the 8-hr. period from 8:00 a.m.—4:00 Pw,
using the fluorimetric method of von Euler and
Floding (Acta. Physiol. Scand. 33/Suppl. 118: 57,
1955). All results are given as mean output in
myug/min.+s.e. (Imug = 0.001 ug). The non
smokers excreted 8.0+1.3 myg/min. epinephrine
and 38.1+4.2 myg/min. norepinephrine. The
smokers, who smoked an average of 9.7 cigarette
during the 8-hour period, excreted 8.2+-0.7 myg/
min. epinephrine and 36.6+3.0 myg/min. nor-
epinephrine. The differences in the epinephrine
and norepinephrine output of non-smokers as
compared to smokers are not significantly dif.
ferent. In a second series of experiments 8
determinations of epinephrine-norepinephrine
output were made on 11 students who normally
smoked during a 2-hr. control period in which they
refrained from smoking and during a 2-hr. period
in which they smoked an average of 6.4 cigarettes.
While smoking, the epinephrine output increased
from a control value of 8.2+1.4 myg/min. to
10.2+1.6 myg/min. This mean increase in epi-
nephrine output of 2.0+0.43 myug/min. is sta-
tistically significant. The norepinephrine output
decreased from 32.8+3.3 myug/min. to 29.8+24
myug/min., a change which is not significant.

1616. Response of microscopic circulation to
dextran and polyvinylpyrrolidone follow:
ing hemorrhage during hypothermia. R. A.
Waup. Dept. of Pharmacology, Univ. of Western
Ontario Med. School, London, Canada.

Rabbits were arranged for recording blood
pressure, heart rate, rectal temperature and for
observation and microphotography of the capil-
laries and other small vessels of the sclera. They
were then placed in a cold room at minus 30°C.
At 2 hr., when the rectal temperature was 28°C
the animals were bled to 30 mm Hg and then
transfused with the blood, dextran or polyvinyl-
pyrrolidone. Controls were carried out at normal
room temperature. In the cold there was an initial
rise in blood pressure. The fall in blood pressure
following withdrawal of a given amount of blood
was much greater in the cold room. The rectal
temperature fell to 21°C and the heart rate to 4
to 20 in 3 hr. When the rectal temperature reached

Volume ij

es in thé
ctions of
Kline and

utput ¢
man. J)
of Phar:
|. School,

ne output
nd of Y
termined
1:00 P.M,
juler and
. 118: 57,
utput in
“he non
inephrine
ine. The
‘igarettes
nin. nor-
ine phrine
okers as
ntly dif-
nents 3
nephrine
normally
hich they
r. period
garettes.
increased
/min. to
. in epi
. is sta
e output
29.8+26
ant.

ation to

follow:
ia. R.A
' Western

g blood
and for
he capil-
ra. They
us 30°C.
vas 28°C
nd then
sly vinyl-
t normal
un initial
pressure
of blood
1e rectal
ate to 4
reached

March 1956

98°C many of the capillaries were devoid of blood
and others contained widely separated groups of
cells. The contour of the venules changed and the
distance between them increased. Hemorrhage at
a rectal temperature of 28°C caused the blood to
disappear from more capillaries, while the course
of others and the venules were only partly out-
lined by groups of cells. Reinfusion with blood
in the cold reestablished the circulation in the
capillaries but the blood pressure rose very little.
Following transfusion with dextran, the capillary
circulation continued to deteriorate and clumping
of the cells in the venules increased progressively.
The effects of transfusion with polyvinylpyrroli-
done were similar to those of dextran. (Supported
by Defence Research Board, Canada.)

1617. Comparison of toxic manifestations
produced by three cardiac glycosides,
digitoxin, digoxin, and acetyl digitoxin.
Davip Wax AND JAacoB KIRSHNER (introduced
by Artuur C. DeGrarr). VA Hosp., Kings-
bridge Road, Bronz, N. Y.

The widespread use of the cardiac glycosides
has coincided with an increased incidence of
digitalis intoxication. It has been claimed that
the glycosides differ in their manifestations of
toxicity. During studies to determine the rates of
dissipation of effect and the optimal maintenance
dosage of several of the cardiac glycosides,
patients were. digitalized to minor toxicity with
Digitoxin, Digoxin or acetyl Digitoxin. At each
state of toxicity, the gastrointestinal, cardio-
vascular, and neurologic symptoms were noted.
These symptoms occurred in the above order of
frequency. Digoxin produced gastrointestinal
manifestations most often—alone or associated
with other symptoms. Cardiovascular and neuro-
logic symptoms did not occur as isolated phe-
nomena with digoxin. In contrast to Digoxin and
acetyl Digitoxin, Digitoxin induced _ isolated
cardiovascular toxic manifestations most com-
monly. Acetyl Digitoxin produced gastrointestinal
symptoms slightly more frequently than cardio-
vascular toxicity; however, the latter occurred
more often alone. Nervous system toxicity with
acetyl Digitoxin was more severe than with the 2
other glycosides. Isolated neurological phenomena
were the least common manifestation of toxicity.
Involvement of 3 systems simultaneously was
least frequent with digitoxin. Digitoxin and
Digoxin given successively to 5 patients produced
results similar to that of the over-all group.
Digitoxin produced cardiovascular toxicity most
frequently; and Digoxin, gastrointestinal toxicity.

1618. Effect of hepatotoxins and metabolic
inhibitors upon the biliary excretion of
methoxychlor. JoHN H. WEIKEL, JR. (intro-
duced by Epwin P. Laue). Div. of Pharma-

E XUM

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

497

cology, Food and Drug Admin., Washington,

D.C.

In 1951 Laug and Kunze showed that rat liver
damage as induced by carbon tetrachloride
increased the oral toxicity of methoxychlor as
well as increasing the accumulation in tissues
(Federation Proc. 10: 318, 1951). Unpublished
results from this laboratory suggested that these
effects might be due to a destruction of the de-
toxication mechanism and/or an embarrassment
of the excretory mechanism. We have since shown
that the principal route of detoxication of meth-
oxychlor and excretion of its metabolites involves
the liver-bile circuit. Thus a measure of the
biliary excretion of the metabolites would test the
validity of this suggestion and perhaps give a clue
as to the mechanism whereby ‘methoxychlor is
detoxified and excreted. For these investigations a
series of toxins was used. First, for a generalized
liver necrosis, carbon tetrachloride was ad-
ministered orally. India ink was used as a blockade
of the reticulo-endothelial system; sodium azide
and sodium fluoroacetate were employed to block
energy utilization systems and SKF 525-A was
used to inhibit the liver microsome enzyme
systems. Of these poisons, carbon tetrachloride
was the only one found to reduce significantly the
biliary excretion of methoxychlor metabolites.
Further, the degree of inhibition seems to correlate
better with the inhibition of bile production per se
than with dose or gross liver damage. The failure
of the other inhibitors indicates an independence
of this excretory system from many of the more
common effectors.

1619. Mechanism of action of N-allylnormor-
phine in morphine-induced respiratory
depression in man. HERBERT WENDEL AND
CHRISTIAN J. LAMBERTSEN. Lab. of Pharma-
cology, Univ. of Pennsylvania School of Medicine,
Philadelphia.

Separate and combined effects upon respiration
of morphine sulfate (10 mg/70 kg) and N-allyl-
normorphine hydrochloride (10 mg/70 kg) were
studied in 15 volunteers. The method consisted
of relating respiratory minute volume to corre-
sponding alveolar pCO, at various inspired CO,
concentrations. Drug effects were measured as
changes in alveolar pCO, at selected RMV levels
before and after drug administration. N-allyl-
normorphine was found to be less than two-thirds
as potent in terms of mg of salt injected, and half
as effective in terms of moles of pure base. N-allyl-
normorphine altered morphine depression sig-
nificantly differently among subjects, both
synergism and antagonism being observed, but on
the average the combined effect of the 2 drugs
was not significantly different from that of mor-
phine alone. After combined administration it was
found that 88% of the N-allylnormorphine effect

498

substituted for 76% of the morphine effect. This,
in conjunction with the agreement between the
ratio of

76 % morphine effect replaced
88 \ % N-allylnormorphine effect substituted

and that of the molecular weights of the 2 drugs,
strongly suggests that in their combined action
N-allylnormorphine competes with morphine on
an equimolecular basis. Antagonistic and syner-
gistic effectiveness of N-allyinormorphine de-
pended upon the ratio of the separate effects of
the 2 drugs. A ‘separate effect’ ratio of 0.56 or
NANM
Morphine
tagonistic action of N-allylnormorphine, and a
ratio of 0.88 or greater a significant synergistic
action. Corresponding dose ratios for

NANM-HCL
Morphine- H.SO,

are 0.78 and 1.23, respectively.

smaller for defined a significant an-

1620. Biogenesis of atropine. F. R. Wrest anp
E. 8. Mrxa (introduced by E. M. K. Gerine).
Dept. of Pharmacology, Univ. of Chicago, Chicago,
Til.

The site of synthesis and the biogenesis of
atropine in Atropa belladonna are being investi-
gated utilizing in vitro and in vivo techniques.
Atropine was obtained from sterile root cultures
grown for 6 months after 3 successive transfers,
while none was found in the stems grown under
similar conditions. The in vivo approach involved
infiltration of certain stable and radioactive
compounds into the leaves of intact plants. During
the experimental period, plants were kept in a
gas-tight enclosure enabling determination of
carbon dioxide. Radioactive atropine was ob-
tained from plants into which C™ glucose had been
infiltrated. Approximately 0.015% of the glucose
activity was present in the crystalline atropine
and 1.7% appeared as radioactive carbon dioxide.
C™ glucose is incorporated most rapidly in the
young, actively growing leaf tissues. Under the
above conditions 5 mg/day of glucose can be
incorporated into the plant. Preliminary studies
indicate that the atropine moiety might be
synthesized in organs other than the root provided
that the necessary precursors and an adequate
supply of certain carbohydrates are available. The
role of certain suspected intermediates in the bio-
genesis of atropine is currently being studied.

1621. Cardiac actions of epinephrine and
levarterenol in the intact dog. T. C. West
AND R. F. Rusumer. Depts. of Pharmacology
and of Physiology and Biophysics, School of
Medicine, Univ. of Washington, Seattle.

FEDERATION PROCEEDINGS

Volume 16

Cardiac responses to epinephrine and levar-
terenol were studied in intact dogs surgically
prepared under aseptic conditions for direct
recording of left ventricular pressure and left
ventricular circumference. It was possible to
observe drug effects in the conscious dog and to
compare results with those obtained under pento-
barbital anesthesia. From the direct measure-
ments other parameters were derived (e.g., heart
rate, stroke work, etc.). Intravenous drug infusion
at desired rates was effected by means of an
infusion pump connected to a_ polyethylene
catheter inserted into a superficial antecubital
vein. Concentrations of epinephrine and _levar-
terenol were matched on a molar basis, using a
range of concentration varying from 1 X 10-9
M/kg/min. to 4 X 10-9 M/kg/min. The response
from dog to dog at a given concentration often
varied greatly. This was predominantly a quanti-
tative variation related to concentration, so that
higher dose levels tended to yield more uniform
results. In general, the data support the following
statements: in the conscious animal; a) levar-
terenol retarded rate, increased ventricular
systolic and diastolic pressures, increased stroke
work and cumulative work, increased ventricular
diastolic circumference and stroke circumference
change with little effect on systolic circumference;
b) epinephrine at equimolar concentrations
generally induced changes directionally similar
to those resulting from levarterenol but of lesser
magnitude. Anesthesia generally influenced the
results quantitatively in a manner expected to
result from depression of homeostatic mechanisms.
Exception to and extension of these generalities
will be discussed. (Supported in part by grants
from the Natl. Heart Inst., PHS.)

1622. Effects of digitalis on glycogen frac-
tions of the heart. B. A. WESTFALL AND F,
A. Purpy.* Dept. of Physiology and Pharma-
cology, Univ. of Missouri School of Medicine,
Columbia.

The effects of digitalis glycosides on the levels
of glycogen fractions in the heart of the albino rat
were determined. One ml of an aqueous infusion
of digitalis per 100 gm of body weight was injected
intraperitoneally into adult rats. One tenth of a
ml of this infusion was equivalent to .0005 mg of
ouabain in frogs. Twelve rats were used to estab-
lish each point in the control and in the experi-
mental animals 12 hr., 24 hr. and 36 hr. after
receiving digitalis. The rats were decapitated
and the hearts removed quickly. A sample of the
heart was obtained, weighed, frozen and _ho-
mogenized at about 3°C. Trichloracetic acid in-
soluble fractions and TCA soluble fractions of
glycogen were determined, and the values ex-
pressed in mg of glycogen per 100 gm of heart

me 16

levar-
ically
lirect
| left
le to
nd to
ento-
sure-
heart
usion
of an
ylene
bital
evar-
ing a
10-°
onse
often
anti-
that
form
wing
evar-
cular
broke
cular
rence
ence;
tions
nilar
esser
- the
d to
isms,
lities
rants

rac-
DF.
rma-
cine,

evels
o rat
ision
cted
of a
ig of
stab-
peri-
after
ated
f the

ho-
1 in-
is of
: eX-
neart

March 1956

tissue. There was a significant difference between
the levels of the TCA insoluble fraction in the
control (168) and in the experimental animals 24
hr. (118, P < .002) and 36 hr. (101, P < .001) after
receiving digitalis. Furthermore, there was a
greater difference between the levels of the TCA
soluble fractions of the controls (132) and the
experimental animals 24 hr. (14, P < .001) and
36 hr. (26, P < .001) after digitalis. (Supported
by a grant from the U. S. Department of Health,
Education and Welfare.)

1623. Anesthesia. XLVIX: hypnosis with
analgesic combinations. JosEPpH M. WuitTe,*
CuarRE K. Hetsse* AND JOHN C. KRaAntTz, JR.
Dept. of Pharmacology, Univ. of Maryland
School of Medicine, Baltimore.

Recently Berger demonstrated an unusual type
of hypnotic action elicited by mixtures of salicyl-
amide and acetophenetidin when injected intra-
peritoneally in acacia suspension into mice. He
observed that levels of the individual analgesics
well below those which produced no hypnosis,
when given simultaneously elicited a hypnotic
action of considerable duration. We became
interested in the induction of hypnosis by salicyl-
amide combinations and decided to extend the
study to other compounds and to test the effect
on other species including man. Several pre-
liminary doses were employed for probing pur-
poses. The 280 mg/kg dose of salicylamide was
selected as a basis for comparative study. The
compounds studied were salicylamide, aceto-
phenetidin, acetanilid, acetoparaminophenol
(APAP) and acetylsalicylic acid. Our study
revealed that salicylamide is not unique in pro-
ducing this type of hypnotic response. Unsus-
pectingly we observed that acetanilid enjoys the
same type of action. In non-hypnotic doses with
acetanilid definite hypnosis is elicited with the
simultaneous administration of non-hypnotic
doses of acetophenetidin. It does appear from our
studies that this type of analgesic combination-
hypnosis is characteristic only of those agents
which contain an amino group in the molecule.
We have confirmed the findings of Berger with
respect to the hypnosis elicited by salicylamide-
acetophenetidin combinations. The phenomenon
prevails to a greater or lesser degree with APAP
combined with acetanilid. Further, the hypnotic-
combination effect of salicylamide is evoked by
acetanilid. Studies in man are suggestive of the
evidence of the sedative action of large doses of
salicylamide and APAP combinations.

1624. Cardiac conduction in the dog during
anesthesia with Fluoromar. JosEPH M.
Waits, Jr., F. Nett Nations AND L. STANTON
StavNney (introduced by Lucten E. Morris).

E XUM

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

499

Div. of Anesthesiology, Univ. of Washington,

Seattle.

During anesthesia with chloroform, ethyl
chloride, cyclopropane, and _ trichloroethylene,
severe cardiac irregularities may be initiated by
intravenous administration of epinephrine. The
present study applies the method of Meek, Hatha-
way, and Orth to dogs anesthetized with trifluoro-
ethylvinylether (Fluoromar), and compares the
conduction changes produced with those elicited
during cyclopropane anesthesia in the same
animals. Control records were obtained on 5
healthy adult mongrel dogs prior to anesthesia
subsequent to intravenous administration of
epinephrine .01 mg/kg. The unpremedicated dogs
were then anesthetized with Fluoromar. Nitrous
oxide and oxygen were used to vaporize the liquid
agent. Maintenance was by an endotracheal,
semi-closed to and fro absorption system with gas
flows of 10 liters or more. One week later, the
same dogs were anesthetized with cyclopropane
using an endotracheal closed to and fro absorption
system. Adequate oxygenation and ventilation
were provided at all times. Thirty minutes equili-
bration in surgical anesthesia preceded the
challenge doses of intravenous epinephrine.
Ventricular tachycardia was sought as the end-
point. Lead II was recorded on a direct writing
viso-cardiette at 10-sec. intervals. Ventricular
tachycardia was obtained after the challenge dose
of epinephrine in each dog during cyclopropane
anesthesia. No irregularities were observed conse-
quent upon induction and maintenance of anes-
thesia with Fluoromar. The most severe
arrhythmia subsequent to epinephrine during
Fluoromar anesthesia was an occasional premature
ventricular complex. Ventricular tachycardia was
not demonstrated in any of the 5 dogs even after
administration of twice the challenge dose of
epinephrine.

1625. Synthesis of thio-esters of acylated
glycine; a study of their properties. VIRGIL
D. WiesetHaus, Atice F. Hogans aNnp Davip
C. Titus (introduced by Kart H. Beyer).
Pharmacology Section, Sharp & Dohme, Div. of
Merck & Co., Inc., West Point, Pa.

The CoASH and GSH thio-esters of p-acetyl-
aminohippurate, hippurate and acetylglycine have
been synthesized. The stabilities of these various
derivatives are comparable to the stability of the
acetyl-thio-esters of CoASH and GSH. All of
these derivatives form hydroxamates readily.
Extracts of renal tissue will cleave the thio-esters
of acetate and hippurate and in each case the
glutathione derivatives are split very much more
rapidly than the CoASH derivatives. BeNEMID
(3 X 10-? M) does not inhibit the cleavage of
these esters. Thiotransacetylase activity in the

500 FEDERATION PROCEEDINGS

renal extracts is indicated by the following: If
GSH is added to a reaction tube containing buffer,
hippuryl-S CoA and renal extract, there is an
accelerated rate of loss of hydroxamate-forming
material. BeNEMID does not prevent this
acceleration of cleavage and, therefore, pre-
sumably does not inhibit the thiotransacetylase
activity. We feel, for reasons previously stated
(Federation Proc. 13: 164, 1954), that the renal
tubular secretion of hippurate involves the
transfer of CoASH from acetyl-S CoA to hip-
purate. This reaction should be reversible. We
have attempted to demonstrate this postulated
activity of renal tissues by coupling the hippury1-S
CoA-renal extract system with the malic de-
hydrogenase and citrate condensing enzyme
reactions. No reaction occurs if either hippuryl-S
CoA or GSH are omitted. When both are present
there occurs an apparent reduction of DPN, but
in poor yield. If hippuryl-SG is employed instead
of the CoASH ester no reaction whatever occurs.
Further analyses and fractionation of renal tissue
preparations are required to characterize the
above described system.

1626. Antagonists to neuro-muscular block
produced by Sarin. J. H. Wiis, ANNE M.
KUNKEL* AND JEAN S. Monter.* Pharmacology
Branch, Chem. Corps Med. Labs.

Compounds have been examined for effective-
ness in overcoming neuro-muscular transmission
block established by a P-containing cholinesterase
inhibitor (Sarin). Atropine had almost no activity;
N-methyl and N-isopropyl atropinium salts had
temporary effects only. Other quaternary com-
pounds, including Lergigan methiodide, Dibuto-
line, and N-benzyl and N-phenacyl atropinium
salts, had more pronounced effects. Also effective
were certain bis (ammonoethy]) and bis (ammono-
propyl) derivatives of oxamide. A monoquater-
nary oxamide derivative had some activity.

1627. Some pharmacological effects of sapo-
nins of alfalfa and ladino clover. RoBERT
H. Wiison, Martin B. SrpEMAN* AND FLoyr
DeEps. Western Utilization Research Branch,
Agricultural Research Service, USDA, Albany,
Calif.

The saponins studied were a water-soluble
mixture from alfalfa and a crystalline saponin
from both alfalfa and ladino clover. Acute oral,
intraperitoneal, and intravenous toxicities in
mice were much greater for the water-soluble
saponin than for the crystalline saponin. Sub-
acute oral toxicity was not apparent in rats,
guinea pigs, and rabbits after 15 days on diets
containing up to 2% saponin. On isolated strips
from rabbit intestine and beef rumen, both the
water-soluble alfalfa and the crystalline saponins

Volume 16

produced qualitatively similar reactions, with the
crystalline saponin the more effective. On first
application there was an immediate decrease in
peristalsis, shortly followed by a temporary
increase in tonus. Subsequent applications pro-
duced progressively less increase in tonus, and
ultimately only decreased tonus was apparent,
Doses of saponin too small to affect normal
contraction still modified the tissue so that the
typical action of first application was not obtained
with subsequent effective dosages, and only
decrease in tonus was obtained. Reaction of strips
to alfalfa saponin differed qualitatively from
reactions to alfalfa juice and to commercial
(yucca?) saponin. Hemolytic activities of various
saponins differ greatly. The crystalline ladino
clover saponin did not hemolyze red cells; the
water-soluble alfalfa saponin hemolyzed but not
as readily as did commercial saponin. Hemolysis
was observed after parenteral administration of
alfalfa saponin but not after oral dosing. The
hemolytic activities of the saponins were not
found to be correlated with reduction in surface
tension.

1628. Effect of some antihistaminic agents
on experimental atrial flutter. Martin M.
WIinBuRY AND Barpara L. Atwortu.* Re-
search Div., Schering Corp., Bloomfield, N. J.
Several antihistaminic agents in current clinical

use were studied for atrial antiarrhythmic activity

in morphine-nembutalized open chest dogs. An
atrial arrhythmia (flutter or fibrillation) was
induced either by crush and stimulation of the
intervenous bridge (ROSENBLEUTH AND Garcia

Ramos, Arch. Inst. Cardiol. Mexico, 17: 1, 1947) or

by the application of aconitine (ScHERF, Proc.

Soc. Exper. Biol. & Med. 64: 238, 1947). The

antihistaminic agents were administered in in-

creasing dosage until there was reversion to sinus
rhythm or side effects precluded further treat-
ment. The compounds studied can be classified as
aromatic derivatives of: 1) ethylenediamine;
2) aminoethylether; 3) propylamine and 4)
phenothiazine-ethylamine. Compounds more ac-
tive than quinidine on a weight basis were:
pyrilamine (Neoantergan); methapyrilene (Then-
ylene); thonzylamine (Neohetramine); diphen-
hydramine (Benadryl) chlorprophenpyridamine

(Chlor-Trimeton) and phenindamine (Thephorin).

Compounds with antiarrhythmic activity equal to

quinidine were: tripelennamine (Pyribenzamine),

doxylamine (Decapryn) and promethazine (Phen-
ergan). Diethazine (Diparcol) was less active than
quinidine while methaphenilene (Diatrine) pro-
phenpyridamine (Trimeton) and chlorpromazine

(Thorazine) were relatively inactive. Thus in the

ethylenediamine series N-(2-pyridyl) or N-(2-

pyrimidyl) derivatives (Pyribenzamine, Neo-

sas & 4 fA A.

E XUM

me 16

th the
n first
ase in
porary
S pro-
3, and
arent,
ormal
at the
tained

only
strips

from
1ercial
‘arious
ladino
Is; the
ut not
10lysis
ion of
;. The
re not
urface

gents
IN M.
* Re-
N.
linical
“tivity
zs. An
) was
of the
TARCIA
947) or
Proce.
). The
in in-
) sinus
treat-
fied as
amine;
nd 4)
re ac-
were:
(Then-
jiphen-
lamine
borin).
qual to
mine),
(Phen-
re than
») pro-
mazine
in the
N-(2-

Neo-

March 1956

antergan, Thenylene and Neohetramine) were
active whereas the N-(phenyl) derivative (Dia-
trine) was inactive. In contrast, in the amino-
ethylether series the phenyl derivative, Benadryl,
was more active than the 2-pyridyl derivative,
Decapryn. Ring substituted chlorine had varying
effects. In the propylamine series, para chlorina-
tion converted the inactive Trimeton to the highly
active Chlor-Trimeton. On the other hand, in the
10-(N-dialkylaminoalkyl) phenothiazine series,
chlorpromazine was inactive while the related
non-chlorinated compounds (Phenergan and
Diparcol) were active.

1629. Influence of rate of stimulation and
fatigue on sensitivity to neuromuscular
blocking agents. L. Wisticki (introduced by
Cart C. PreirreR). Dept. of Pharmacology,
Hebrew Univ.-Hadassah Med. School, Jerusalem,
Israel.

Sensitivity to neuromuscular blocking agents
(n.m.b.a.) differs between various muscle groups.
Factors responsible for these differences were
investigated in cats under pentobarbitone anes-
thesia; carotid B.P., respiration, and isometric
contractions of both m. triceps surae after indirect
stimulation were recorded. Differences in the
response to succinylcholine and d-tubocurarine
occurred even between symmetrical muscles. The
effect of n.m.b.a. during supramaximal stimula-
tion at 10/min. differed hardly from their action
at lower frequencies, but became magnified at
30/min. and still more marked at higher rates.
These frequencies approach physiological/dis-
charge rates in muscle fibers. Muscles with high
discharge rates are particularly sensitive to
n.m.b.a. During subtetanic stimulation recovery
from a small dose of succinylcholine often began
with contractions at frequencies which were lower
than the rate of stimulation; the original sub-
tetanic response developed only within 2 min.
Fatigue intensified, but morphine and meperidine
did not significantly influence the effect to n.m.b.a.
A protracted block by succinylcholine or d-tubo-
curarine, applied during continuous stimulation,
acted on the muscle like a period of rest.

1630. Coronary vasodilator properties of
purine and pyrimidine derivatives. Mary
M. Wour anv Rosert M. Berne (introduced
by Gerorce Sayers). Dept. of Physiology,
Western Reserve Univ. School of Medicine,
Cleveland, Ohio.

Experiments were performed on 28 open-chest
dogs anesthetized with pentobarbital. The
circumflex branch of the left coronary artery was
cannulated and perfused via the subclavian artery
at a constant pressure. Flow was measured by an
optically recording rotameter. Sodium salts of the

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS 501

compounds to be tested were dissolved in 0.9%
NaCl solution and delivered into the tubing to the
coronary cannula at a constant rate of 0.93 ml/
min. Maximal vasodilator effects on the dog
coronary arteries were obtained with infusions of
ATP at rates of 0.2 to 0.3 um/min. Comparison of
the potency of this compound with other members
of the adenine series of nucleotides revealed ADP
to be approximately of equal potency, whereas
AMP and adenosine were about two-thirds as
effective in reducing vascular resistance. Adenine
and all members of the hypoxanthine series with
the exception of ITP lacked vasodilator proper-
ties. ITP produced small increases in coronary
blood flow (CBF). The derivatives of guanine and
cytosine were also inactive. UTP was the only
member of the uracil derivatives which evoked an
increase in CBF and was found to be approxi-
mately one-third as potent as ATP. Although an
increase in myocardial oxygen consumption was
observed during infusion of ATP and UTP, the
elevation in CBF was greater than that necessary
to meet the increased oxygen demand. Therefore
the action of these compounds is believed to be
primarily on the vessels and not secondary to an
increased rate of myocardial metabolism.

1631. Distribution of radiochloride, radio-
sulfate, and inulin in brain of rats. D. M.
Woopsury, P. 8. Timmras, A. Kocnu* anp A.
Bauuarp.* Dept. of Pharmacology, Univ. of
Utah College of Medicine, Salt Lake City.

The distribution of inulin, radiochloride (Cl**),
and radiosulfate (S*5O,) has been studied in the
brain of rats. Inulin was injected into nephrecto-
mized rats and the animals were killed 16 hr.
later. Rats were sacrificed 10 and 30 min. and
1, 2, 4, 8, 16, 24, and 48 hr. after intraperitoneal
injection of 20 ue S*5O, and 0.2 ue of Cl**. Total
chloride space was compared with the correspond-
ing Cl?6, $%60,, and inulin spaces. Brain Cl space
averaged 24.7% of wet weight. Cl** space pro-
gressively increased until, at 8 hr., it equalled the
total Cl space and subsequently remained at this
level. S*5O, space increased to a value of about
4% of wet weight at 4 hr. and remained at this
level for 16 hr., it increased markedly at 24 and
48 hr. to about 32%. Inulin space was found to be
4 to 5% after 16 hr., thus, inulin and S*5O, spaces
are equal up to this time. On the assumption that
$350, and inulin measure the ‘rapidly equili-
brating’ water in brain as they do in muscle, then
this space amounts only to 4% of brain weight. As
in muscle, Cl space in brain is a measure of the
total extracellular space and is composed of a
‘rapidly equilibrating’ space and a ‘slowly equili-
brating’ connective tissue space. Implications of
these results to the problem of the blood-brain
barrier and glial tissue will be discussed. (Sup-

502

ported in part by contract number 18(600)921,
USAF and grant B-381 from the Natl]. Insts. of
Health, PHS.)

1632. Pseudotachyphylaxis. Ropert A. Woop-
BURY AND JOHN W. Wiks.* Div. of Pharma-
cology, Univ. of Tennessee Med. Units,
Memphis.

In dogs intravenous injections of vasopressin
(0.3 units/kg) repeated at 30-min intervals pro-
duce progressively smaller increases in arterial
pressure. This marked reduction in the pressor
response which appears after one or two such
injections has been explained as tachyphylaxis.
Dogs given ouabain (0.04 mg/kg) intravenously
30-min. to one hour prior to any vasopressin
injections, in most instances have not shown this
pronounced reduction in blood pressure response
to vasopressin even though as many as 10 such
injections were made. Dogs given ouabain intra-
venously following 3 such injections of vaso-
pressin, when tachyphylaxis was supposedly
present, have shown blood pressure responses to
subsequent injections of vasopressin which were
essentially similar to those caused by the first
vasopressin injection. These data suggest that
tachyphylaxis to vasopressin develops less readily
than previously stated and that inability of the
heart to pump blood against elevated pressures
results in an apparent tachyphylaxis or ‘pseudo-
tachyphylaxis’. Vasopressin injected as above
reduces or eliminates the reserve capacity of the
heart to do work, presumably by coronary con-
striction. Ouabain by increasing the force of
cardiac contractions removes or reduces this
limiting factor, thereby reestablishing the pressor
response to vasopressin. Dogs previously digi-
talized should serve as better test animals for the
bioassay of vasopressin.

1633. Comparative distribution of morphine
and nalorphine in dog brain. L. A. Woops.
Dept. of Pharmacology, Univ. of Michigan, Ann
Arbor.

The method for the estimation of morphine in
tissue (Wdons et al. J. Pharmacol. & Exper.
Therap. 111: 64, 1954) was modified for the de-
termination of free morphine or nalorphine in
brain tissue in concentrations of one wg/gm and
above. Each of 25 mongrel dogs was injected
subcutaneously with the selected dose (calculated
as free base) of morphine or nalorphine, or both.
After the desired interval the dog was anesthetized
with thiopental, exsanguinated and the head
perfused with physiological saline. The brain was
removed and frozen promptly on solid carbon
dioxide. The mean values (and range) in »g/gm
for the concentration of free morphine or nalor-
phine in the brain of 2 animals, unless otherwise

FEDERATION PROCEEDINGS

Volume 16

indicated, at the stated intervals after the ad-
ministration of drug were as follows: 30 mg/kg of
morphine or nalorphine—90 min.; morphine, 4.3
(3.7-5.6) (6 dogs); nalorphine, 14 (10-18) (6 dogs);
4 hr.; morphine, 4.6 (4.1-5.1); nalorphine, 2.6
(1.9-3.3), 8 hr.; morphine, 3.8 (3.3-4.2). Thirty
mg/kg of morphine and 3 mg/kg of nalorphine
simu!taneously—90 min.; morphine, 4.2 (3.1-5.0)
(3 dogs). Twenty-two mg/kg of morphine—90
min., morphine, 2.5 (2.3-2.7). Ten mg/kg of
morphine—90 min.; morphine, 1.2 (1.0-1.3).
Therefore nalorphine penetrates dog brain more
rapidly and to a greater extent than morphine.
Similarly nalorphine makes more rapid egress
from brain than does morphine. The administra-
tion of nalorphine with morphine in a ratio of
1:10 does not significantly alter the brain concen-
tration of the latter drug even though most of the
pharmacological actions of morphine are pre-
vented. (Supported in part by grant B-625, Natl.
Insts. of Health, PHS.)

1634. Shock produced in dogs by infusions of
norepinephrine. ALBERT C. YARD* AND
Mark Nickerson. Dept. of Physiology and
Med. Research, Univ. of Manitoba Faculty of
Medicine, Winnipeg, Canada.

Severe prolonged vasoconstriction resulting in
tissue hypoxia has been implicated in the etiology
of several types of shock. In an attempt to evalu-
ate the role of such vasoconstriction, solutions of
l-norepinephrine were infused at a constant rate
for 4 hr. into dogs under light pentobarbital
anesthesia. Doses as small as 2.0 yug/kg/min.
given intravenously produced a high mortality.
In most animals the blood pressure increased
initially, but by the end of the infusion period, it
had stabilized near the normal range. Despite this
approximately normal blood pressure, cardiac
output may be greatly reduced during the period
of infusion. Of 11 dogs infused with 2.0 ug/kg/min.,
only 1 survived permanently, 9 died within 60 hr.
and 1 at 108 hr. after the end of the infusion. Those
surviving longer than 12-18 hr. regained conscious-
ness and were usually able to stand and to drink
water for a period of time prior to death. Out-
standing postmortem findings were the presence
of blood in the lumen of the small intestine and
colon, edema of the mucosa of the small intestine
and extensive hemorrhagic areas visible on the
surface of the lung.

1635. Hypotensive action of 1-phenyl-2-di-
methylaminoethoxymethyl-tetrazole _hy-
drochloride (TT-209). G. K. W. Yim,*
E. G. Gross anp H. H. Keasuine. Dept. of
Pharmacology, College of Medicine, State Univ.
Towa, Iowa City.

TT-209 in doses of 5-10 mg/kg i.v. in dogs

er es Se.|h—lULelU lV ee ee

—

ing in
iology
evalu-
ons of
it rate
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. Those
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1-2-di-
» hhy-

Yrm,*
Dept. of
e Univ.

in dogs

March 1956

anesthetized with pentobarbital sodium produces
a fall in blood pressure of 60-80% which returns to
pre-injection levels within 3-2 hr. Responses to
epinephrine or carotid occlusion are not affected.
A second i.v. injection of the same dose is either
ineffective or much less effective. S.G. or i.m.
injections of 10 mg/kg in dogs which have not
previously received TT-209 do not lower blood
pressure. In dogs anesthetized with chloralose,
2.5 mg/kg i.v. usually produces a fall in blood
pressure of about 60% which returns to control
levels in 15-30 min. An hour later, 5 mg/kg usually
produces a fall of about 45% which returns to
control in 15-30 min. Subsequently 10 mg/kg
produces a fall of about 60% which returns to
control in 15-30 min. The hypotensive phase may
be accompanied initially by bradycardia or no
change in heart rate. At the peak of the blood
pressure fall, bradycardia, no change or tachy-
cardia may be observed. Apnea followed by
hyperpnea occurs occasionally or only hyperpnea
may be seen. Walton strain gauge records of heart
contractile force in open-chest dogs reveal a
marked decrease in contractile force following 5
mg/kg TT-209 i.v. Bilateral vagal section di-
minishes this depressant effect on the contractile
force. Further cardiovascular actions of TT-209
will be discussed.

1636. Cure of Trypanosoma equiperdum in-
fection in mice with Netropsin and with
blood fractions. ATHANASIUS E. J. Yoo,*
Joe B. Nasn* anp G. A. Emerson. Univ. of
Texas Med. Branch, Galveston.

Mice treated intravenously or intraperitoneally
with Netropsin sulfate, at a time when their blood
contained >100 trypanosomes per high-power
field, are cleared of their parasitemia. Repeated
examination of their blood was made without
evidence of relapse. According to the dose used, a
single injection or 2 injections on consecutive
days are adequate for cure. Permanent cure is not
usually obtained with arsenical drugs. Blood and
blood fractions from different species also may
effect a permanent cure. Among 80 other agents
tested in this infection, none has potency com-
parable to that of Netropsin sulfate.

1637. Potentiation, in man, of the cardio-
vascular response to epinephrine by di-
phenydramine hydrochloride (benadryl).
Howarp L. ZaupEeR, Georce A. Mora.es*
AND Louis R. Orkin. Dept. of Anesthesiology,
Albert Einstein College of Medicine, Yeshiva
Univ., New York City.

Intravenous injection of small doses of epi-
nephrine into unanesthetized subjects produced
one of two responses on the cardiovascular system.
An increase in pulse rate as well as systolic and

PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

503

diastolic blood pressure was observed most fre-
quently. Benadryl in doses of 50 mg intravenously
potentiated this response to 5.0, 7.5 and 10.0 ug
of epinephrine. With 5 ug the change in pulse
rate was increased 100%, systolic blood pressure
81% and diastolic blood pressure 275%. The
duration of the response was prolonged by 44%.
Benadryl] potentiated larger doses of epinephrine
in a similar fashion. There was also a dose-response
effect of Benadryl. On occasion ventricular pre-
mature contractions occurred at the 10 ug level
following Benadryl. Such disturbances in rhythm
were not seen with epinephrine alone. Several
subjects exhibited a reflex slowing of the pulse
with epinephrine. Occasionally a decrease in
systolic and/or diastolic pressure followed.
Benadryl increased this reflex activity in several
instances. In others the reflex activity was di-
minished and the response converted to a pressor
one. Large doses of atropine had no significant
effect on either the response to epinephrine or its
potentiation by Benadryl. This would indicate
that this action of Benadryl is independent of any
parasympatholytic properties and in all proba-
bility represents an altered response to epi-
nephrine.

1638. Preparation and biological evaluation
of fluorine derivatives of estradiol. Wot-
FRAM ZILLIG AND GERALD C. MUELLER (in-
troduced by F. E. Suarpeman). McArdle Me-
mortal Lab., Med. School, Univ. of Wisconsin,
Madison.

Recent studies in this laboratory have been
concerned with the nature of the interaction of
the estrogenic hormones with tissue constituents
in the process of exerting their biological effects.
In attempting to evaluate the role of the phenolic
A-ring for estrogenic action the 2 and 4 mono-
fluoro derivatives of estradiol have been synthe-
sized by a new method involving the photode-
composition with ultraviolet light of the
corresponding diazonium salts in a solution of
anhydrous hydrogen fluoride and peroxide free
dioxane. After demethylation the chromato-
graphically pure products were assayed for
biological activity. The 4-fluoro estradiol ex-
hibited approximately 40% of the biological
potency of estradiol-178 in the mouse uterine
growth test when compared at levels of 0.1 y of
each compound; the 2-fluoro estradiol showed no
activity at this level and 100 y of this compound
gave a response equivalent to .04 y of estradiol.
Both compounds assayed like impeded estrogens
in the classification of Huggins and Jensen (J.
Exptl. Med. 102: 335, 1955). In addition the 2-fluoro
compound was found to be an effective antagonist
against the promotion of uterine growth by
estradiol-178; 10 y of 2-fluoro estradiol plus 0.1 y

504 FEDERATION PROCEEDINGS Volume 16

of estradiol-178 gave approximately 10% of
uterine weight increase obtained with 0.1 y of
estradiol-178 alone. The fluorinated estradiols
have been made radioactive by conversion to the
corresponding 17 methyl-C' compounds. The
effect of blocking the 2 and 4 positions with

fluorine as well as blocking the 1 position with a
methyl group on the metabolism of the steroid
nucleus in the in vitro metabolic system of Riegel
and Mueller which gives rise to a protein-bound
derivative of radioactive estradiol and at least 4
new metabolites will be reported.

LEE

4-5

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ast 4

AMERICAN SOCIETY FOR EXPERIMENTAL
PATHOLOGY

Forty-first Annual Meeting
Atiantic City, New Jersey, Aprit 16-20, 1956

An asterisk* following an author’s name indicates “by invitation”

1639. Interaction of beef liver glutamic de-
hydrogenase with metal binding agents.
§. J. ADELSTEIN AND B. L. VALLEE (introduced
by Jamzes P. O’HareE). Biophysics Research Lab.
of the Dept. of Medicine, Harvard Med. School
and Peter Bent Brigham Hosp., Boston, and the
Dept. of Biology, Massachusetts Inst. of Tech-
nology, Cambridge.

Crystalline beef liver glutamic dehydrogenase
has been shown to be a zinc metalloenzyme (VAL-
Lee, B. L., S. J. ADELSTEIN AND R. Ouson, J.
Am, Chem. Soc. 77: 5196, 1955). By spectrographic
and microchemical analysis, the enzyme contains
4-5 gm atoms of zinc/m of protein based on a
molecular weight of 10°. As the activity/zinc ratio
reaches a maximum value with progressive puri-
fication and crystallization, there is an aggrega-
tion of zinc and virtual disappearance of all other
metals. Zine is firmly bound to this relatively
heat stable enzyme. The oxidation and reduction
of DPNH and DPN is inhibited by incubating
the enzyme with a number of metal binding
agents: Na2S, BAL, azide, orthophenanthroline
OP), thiourea, diethyldithiocarbamate NaDDC),
tetraethylthiuram disulfide (Antabuse), 8-hy-
droxyquinoline and ammonium N-nitrosophenyl-
hydroxylamine (cupferron). In several instances
the inhibition has been prevented by the addition
of DPN prior to incubation of the enzyme with
these reagents. In the presence of a large excess of
complexing agent, the initial rate of inhibition is
found to be first order with respect to enzyme
concentration. This reaction rate has been studied
both as a function of temperature and inhibitor
concentration. The concentration dependence of
the inhibition by various complexing agents has
also been investigated at equilibrium.

1440. Postparabiotic disappearance of classi-
cal homologous tissue reactions to muscu-
lofascial cross-transplants in rabbits.
RicHarpD ANDRESEN,* CLARENCE MOoNnrRogE,*
DorotHy MappEN* AaNnp GrorGE Hass. Rush
Labs. of Pathology and Surgical Research, Pres-
byterian Hosp., Chicago, Ill.

505

Twenty-five pairs of rabbits were joined in
parabiosis by uniting ears for 13-15 days. All were
then separated and 13 pairs after 10-21 days were
reunited in parabiosis for 5-15 days and then again
separated. Six to 90 days following separation
small cuboid masses of erector spinae muscle with
attached fascia were cross-transplanted between
respective parabionts. Two weeks later, animals
were killed and transplants prepared for micro-
scopic study. This showed 2 types of tissue reac-
tion about equally distributed between the 2
groups of parabionts. The first was characterized
by development of a peculiar angiomatous network
of patent vascular channels beneath the fascia
of the transplant. In spite of intense vasculariza-
tion, there was negligible fibroblastic prolifera-
tion, collagen formation or inflammatory cell
infiltration. The basic architecture of the trans-
plant was unaltered. The second type of response
resembled the first, except for absence of vascular
penetration of the transplant by the host’s tissue.
These characteristic unique reactions were elicited
by homografts made between parabionts as long
as 90 days after separation. They were quite differ-
ent from customary reactions to autologous and
homologous musculofascial transplants in rabbits
not subjected to parabiosis and also from reactions
to homologous transplants in rabbits which have
received successive musculofascia! grafts from
the same donor. They resemble reactions to
homologous musculofascial transplants exposed
to low temperatures or x-irradiation in vitro
before implantation.

1641. Renal lesions in mice receiving 600 r
whole body radiation. WiLtit1AamM ANTOPOL
AND Susi GuAuBAcH.* Josen and Helen Yeamans
Levy Fndn., Beth Israel Hosp., New York City.
In the course of our investigations on effects

of whole body x-radiation on 4-10-month-old

C57 Bl mice (from a stock which has a low in-

cidence of infection when challenged with large

doses of cortisone), 9 of the animals survived from

6-13 months after the administration of 600 r.

All of these developed striking lesions in the kid-

506

ney. In animals dying or killed before the 6-month
period, the glomeruli contained large pyknotic
nuclei and there was progressive increase in inter-
capillary material. In those mice which survived
more than 6 months, the glomeruli were nodular
and resembled those of intercapillary glomerulo-
sclerosis; with more extensive involvement the
entire glomerulus was sclerosed. Generalized
arteriosclerosis and cardiac hypertrophy were
present. One mouse irradiated at 6 months of age
died 7 months later and a 2nd mouse irradiated
at 4 months died 12 months later. These mice
revealed necrotic arteries in the kidney and other
regions and in the 2nd mouse the necrotic vessels
were surrounded by inflammatory cells as in peri-
arteritis nodosa. Post-mortem cultures of the
liver, kidney and lung were negative in these mice.
The serial morphologic changes indicate a direct
initial radiation effect on the renal glomerular
apparatus with progressive glomerulosclerosis.
Cardiac hypertrophn and generalized arterio-
sclerosis ensue.

1642. Advantages of TAMe assay for routine
determination of plasma _ prothrombin.
Puyuus M. Arscottr, J. L. KoppeL anp JOHN
H. Ouwin (introduced by Cectt A. KRAKOWER).
Lab. of Surgical Research, Presbyterian Hosp.,
Chicago, Ill.

At the present time there are 3 methods avail-
able for determination of plasma prothrombin.
The 1-stage technique is being used most widely
in clinical work though there is considerable
doubt that it measures only prothrombin. Its
other main disadvantage is the often observed
extreme variability in assay results obtained for
the same plasma when different types of thrombo-
plastic preparations are used. The 2-stage method,
on the other hand, is considered to measure pro-
thrombin more specifically, thus allowing for a
more reliable control of anticoagulant therapy;
it appears, however, to be too difficult technically
for routine clinical use. The recently reported
TAMe assay (GLUECK, SHERRY AND TROLL. Proc.
Soc. Exper. Biol. & Med. 87: 646, 1954) was found
to combine the virtues of both techniques as fol-
lows: In a series of over 500 prothrombin deter-
minations in normal plasmas and those obtained
from patients individually maintained on a variety
(8) of anticoagulant drugs the TAMe assay showed
consistently close agreement with the 2-stage
method. Furthermore, the TAMe assay was found
to yield identical results with 5 different thrombo-
plastic preparations. In addition to its repro-
ducibility, another important advantage of this
technique is its relative technical simplicity. It
would appear that insofar as its reliability and
clinical usefulness are concerned, the TAMe
assay is a satisfactory and desirable substitute

FEDERATION PROCEEDINGS

Volume i

for 1-stage and 2-stage techniques. (Supported
by Med. Research and Develop. Board, Office of
the Surgeon General, Dept. of the Army, under
Contract no. DA-49-007-MD-275.)

1643. Ultraviolet absorption of aortic elastic
tissue. Joun P. AYER AND KatTuoryn HAtgy
(introduced by SHIELDS WARREN). Cancer Re-
search Inst., New England Deaconess Hosp,
Boston, Mass.

The ultraviolet absorption of sections of whole,
formalin-fixed canine aorta, canine aorta purified
of collagen, muscle, cells and fat by the method
of Hass, and water-soluble elastin obtained from
the latter by its solution in formic acid are studied
with the Polaroid ultraviolet color translating
microscope. Using transmitted wavelengths of
280 uw, 263 w and 240 yw, corresponding to visible
colors of blue, green and red, the elastic tissue
has a deep pink color with a sheathing of highly
refractile bluish-white. The collagen and muscle
have a neutral gray-brown color and the nuclei
are pale green. Since the contractive effects of
specific stains is eliminated in this procedure, the
inner laminated structure of the aortic lamellae
is clearly seen. The outermost laminae of con-
centrically contiguous lamellae anastomose freely.
Using different combinations of wavelengths of
transmitted ultraviolet light, the maximum ab-
sorption by elastic tissue is found to occur be
tween 270 and 280 uw. Purified water-insoluble
elastica and water-soluble elastin have the same
wavelength of maximal absorption but the amount
is less than that found in elastic tissue of whole
aorta. Water-soluble elastin tested with a Beck-
man photometer is found to have a specific peak
of absorption at a wavelength of 275 uw which is
absent in results for collagen and gelatin.

1644. Alloxan diabetes and cortisone as modi-
fying factors in experimental mucormycosis
(Rhizopus infection). Roger D. Baker, R.
A. ScHoFIELD,* T. Davip ELpER* AND ANGE
P. Spoto, Jr.* Dept. of Pathology, Duke Univ.
School of Medicine and VA Hosp., Durham, N.€.
Intratracheal inoculation of suspensions of

spores of Rhizopus arrhizus into rabbits in the

acute toxic phase (first 3 days) of alloxan diabetes
results in great proliferation of the hyphal form
of the fungus with ulcerative bronchitis, extensive
pneumonia and vascular thrombosis. These ful-
minating features are not observed in rabbits it
the chronic phase of alloxan diabetes or in not

diabetic controls. Similarly, the chronic stage d

alloxan-induced experimental diabetes in mict

does not alter the reaction to intraperitoneal o

intracerebral inoculation of the fungus. Hyper

glycemia in itself does not appear to increase the
host susceptibility of mouse or rabbit to this

ral

ot]

tis:

mi:

164

J ™ CO — a

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E XUM

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KER, R.
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ke Univ.
m, N.C.
sions of
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March 1956

experimental infection. Experimental peritoneal
mucormycosis (Rhizopus oryzae infection) in cor-
tisone-treated mice, rats and guinea pigs spreads
to extraperitoneal organs to a much greater ex-
tent than in normal controls. The lesions of mu-
cormycosis are larger, and there are more of the
hyphal forms of the fungus, in the cortisone-
treated than in the normal animals. Cortisone has
a tendency to promote spreading of this fun-
gous organism in the animal body. (Aided by
Grant E-861 of the Natl. Microbiological Inst.,
Natl. Insts. of Health, PHS.)

1645. Kidney localizing proteins derived from
normal tissues. WILLIAM F. Baz, Irvine L.
Spar AND Rutu L. Goop.uanp (introduced by
GeorGE H. Wurppe). Dept. of Radiation Biol-
ogy, Univ. of Rochester School of Medicine, Roch-
ester, N. Y.

From several tissues of presumably normal ani-
mals, protein fractions have been isolated and
labeled with I! that gave substantial kidney
localization of I'*! following intravenous injection
into animals. One such fraction was obtained by
eluting washed rabbit kidney homogenate at
37°C and pu 3.2. After iodination, the labeled
protein with affinity for kidney was selectively
purified by in vitro absorption on kidney tissue
followed by a second elution. Three days after
this final product was injected into rabbits some
15% was present in the perfused kidneys of these
animals. In a second type of experiment, protein
from a saline extract made at 37°C of minced
rabbit skin was iodinated and a component with
affinity for kidney selectively separated from
other I'*!-labeled material by in vivo concentration
on rat kidney followed, after killing, by elution
of the washed homogenized kidney residue at
0°C and pu 11. The I'*!-labeled protein fraction
rendered soluble by this treatment and injected
intravenously into rats was present in kidneys at
a level of 12.5% of the injected dose 3 days later.
Fractions with appreciable kidney-localizing
tendency have been prepared from other rabbit
and guinea pig tissues. This tendency for some
proteins derived from normal tissues to remain
localized in the kidney may be a factor in kidney
damage associated with traumatic injury to other
tissues. (Supported by the U. S. Army Surgeon
General’s Office and the Atomic Energy Com-
mission.)

1646. An enzyme in mast cells with some
properties resembling chymotrypsin. EARL
P. Benpitt. Dept. of Pathology, Univ. of Chicago
and the LaRabida Jackson Park Sanatorium,
Chicago, Ill.

The chloroacetyl ester of 2-OH-3 naphthoic
acid anilide (CANAS) has been shown (Gomokrt.

E XUM

AMERICAN SOCIETY FOR EXPERIMENTAL PATHOLOGY

507

J. Histochem. & Cytochem. 1: 469, 1953) to serve as
a histochemical substrate for an enzyme present
in mast cells. It is known that chymotrypsin will
split esters of amino acids and certaid chloro-
analogues at rates even greater than the corre-
sponding amides or peptides. For this and other
reasons we were induced to test the activity of
crystallized chymotrypsin on this substrate and
to compare it with the activity of the mast cell
enzyme. For measurement of enzyme activity in
the test tube the histochemical substrate solution
was used after filtration. Spontaneous decomposi-
tion of substrate over periods up to 20 min. in-
cubation at 25°C were accounted for by. appro-
priate blanks. Color was read at 540 mu. Net color
development was linear with time at a given en-
zyme concentration and with enzyme concentra-
tion under appropriate conditions. Mast cell
enzyme was derived from 2 sources: a homogenate
of rat feet skin which has been found to have a
high mast cell content and purified mast cells
isolated from the peritoneal cavity of rats. In
the system used 6.5 mm? of mast cells were found
to have activity equivalent to about 80 ug of
chymotrypsin. The activities of homogenate and
chymotrypsin were 90% inhibited by preincuba-
tion with 10-4 m of DFP. In tissue sections ac-
tivity was similarly inhibited. (Supported in
part by Grant No. H-2141 from the Natl. Heart
Inst., Natl. Insts. of Health, PHS.)

1647. Production of concrements and of bili-
ary tract lesions in rabbits fed 38-choles-
tanol. M. Brevans, E. H. Mossacn* anp R.
Kapuan.* Columbia Univ. Research Div., Gold-
water Memorial Hosp., and Dept. of Medicine,
Columbia Univ., New York City.

Rabbits maintained for 8-23 days on a diet
containing 0.25% or 1% added 36-cholestanol
developed cholelithiasis, cloudy bile and inflam-
matory lesions of the gallbladder and large
bile ducts. No lesions of the biliary tract were
found in control rabbits receiving the stock diet,
or stock diet plus 1% cholesterol or 1% sitosterol.
The addition of 12% triolein to the diet containing
38-cholestanol increased the amount of lipid in
the liver but did not increase the severity of the
lesions of the biliary tract. In rabbits killed at 8
days the bile was cloudy, thick, bluish-green and
contained small concrements. After 3 wk. the con-
crements frequently had coalesced, forming a
cast within the fundus. Concrements were visible
in the intrahepatic ducts. Edema and infiltration
with lymphocytes and histiocytes of the lamina
propria of the gallbladder present in rabbits killed
after 8 days of 36-cholestanol administration were
only slightly increased in animals examined after
23 days of feeding. The changes produced by the
diet containing 1% 38-cholestanol were in general

508

more severe than those seen in rabbits receiving
0.25% 38-cholestanol. No lipid infiltration or
atheromatous plaques in the aorta were found in
these short-term experiments nor was jaundice
observed. Cook, Kliman and Fieser reported
murky bile or gallstones in 4 rabbits fed 38-
cholestanol but did not describe histologic
changes.

1648. Hypertension and renal disease in Wis-
tar rats following 1000 r anoxic total body
irradiation. M.S. Bituines,* L. R. BEnNertT*
AND B. G. Lamson. Depts. of Radiology and
Pathology and Atomic Energy Project, Univ.
of California at Los Angeles School of Medicine,
Los Angeles.

Female Wistar rats receiving 1000 r of total body
radiation delivered under conditions of 5% hy-
poxia at age 4 montis have been observed clinically
for evidence of hypertension during the latter
half of their 24-month postirradiation survival
period. The mean maximum systolic blood pres-
sure of 56 irradiated rats during this period was
195 mm Hg, whereas the mean maximum pressure
of 40 nonirradiated rats has been 1388 mm Hg
during an observation period now extending to
28 months. Twenty-nine of 65 irradiated rats have
developed renal disease characterized histo-
logically by deposition of PAS-positive material
in glomeruli and dilatation of tubules. Mean
maximum blood pressure in those rats with this
renal lesion was 205 mm Hg compared to 187 mm
Hg in those irradiated rats without this renal
change. Kidneys of irradiated and nonirradiated
rats of the same age are approximately equal in
size measured as a percentage of total body weight.
Kidneys of irradiated rats showing histologic renal
disease are not smaller than irradiated kidneys
showing no histologic changes. We have no evi-
dence that hypertension in these irradiated ani-
mals is related to scarred, small contracted kid-
neys. (Based on work performed under Contract
No. AT-04-GEN-12 between the Atomic Energy
Commission and the Univ. of California at Los
Angeles Méd. School.)

1649. Effect of somatotrophic hormone and
chlortetracycline on weight and mortality
of irradiated rats. J. M. B. BLoopwortu, JR.,
Puytuis Arscotr, GrorceE J. HamMwi AND
JosePpH L. Morton (introduced by E. von
Haam). Depts. of Pathology, Radiology and Medi-
cine, Ohio State Univ. College of Medicine, Colum-
bus, Ohio.

Previous work has shown that somatotrophic
hormone (STH) in association with oxytetra-
eycline will decrease the weight loss following
total body irradiation. Rats weighing 200 gm
received 650 r total body irradiation. Daily ad-

FEDERATION PROCEEDINGS

Volume 1§

ministration of 1.5 mg STH parenterally or 10 mg
chlortetracycline in the drinking water was begun
on the day of irradation. Irradiation produced a
rapid and severe weight loss which continued for
4-6 days regardless of therapy. Animals which
subsequently died continued to show rapid weight
loss regardless of therapy. Surviving animals
resumed weight gain 4-6 days after irradiation
regardless of therapy. Once weight gain was re-
sumed STH increased the rate; chlortetracycline
produced no effect. In the first experiment 14-day
mortality of irradiated controls was 100% while
irradiated animals receiving STH showed a sta-
tistically insignificant decrease in mortality,
There was no sex difference. Chlortetracycline
reduced the mortality to 27%. In the second ex-
periment 21-day mortality of irradiated controls
was 75%. STH increased the mortality to 100%,
as well as increasing the rapidity of death. Chlor-
tetracycline decreased the mortality to 42%.
These findings show that administration of STH
during the catabolic period immediately following
irradiation increases the mortality but, if the
animal survives, the initial catabolic period subse-
quent weight gain is enhanced. Chlortetracylcine
decreases the mortality following irradiation but
has no effect on the weight of the animals,

1650. Factors regulating lymph formation.
J. L. Botuman. Mayo Clinic, Rochester, Minn.
It has been shown that the intestinal adminis-

tration of sodium chloride in physiological
amounts produces a much larger volume of intes-
tinal lymph than does administration of equal
volumes of water with any other solute. With the
standard procedure we have used, rats receiving
55 ml of water by intestine in 24 hr. secrete about
40 ml of urine and 15 ml of intestinal lymph from
the cannulated lymphatic. If sodium chloride is
included in the fluid administered, only 15 ml of
urine is obtained and 40 ml of intestinal lymph.
Both the water and the sodium of the lymph in
either case is derived from the blood and only
traces may derive directly from intestinal absorp-
tion. Administration of similar volume by periph-
eral vein produces lymph and urine similar to
water by intestine even if the venous injection
contains sodium chloride. However, administra-
tion by portal vein produces similar lymph
and urine changes as was found with intestinal
administration. If urine secretion is prevented by
ligation of both ureters portal injection of saline
produces larger amounts of lymph than similar
amounts of other solutions. Adrenalectomized
animals form larger amounts of lymph and small
amounts of urine from aqueous injections whether
or not sodium chloride has been added. Pitressin
administration also increases the lymph flow from
water administration.

a Oa ee ee a, ae ee a a

ume 16

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March 1956

1651. Hépatitis in hamsters inoculated with
equine abortion virus. II. Weight, protein,
and nucleic acid determinations of isolated
nuclei. E. C, BRacKEN* AND CHARLES C. Ran-
DALL. Dept. of Microbiology, Vanderbilt Univ.
School of Medicine, Nashville, Tenn.

Livers of young hamsters, infected with equine
abortion virus, were collected over a period of 15
hr. at 3-hr. intervals, and in some cases from ani-
mals in terminal stages of infection. All samples
were represented by pooled livers of at least 5
hamsters. Controls consisted of livers from unin-
oculated hamsters and from hamsters injected
with suspensions of normal liver and killed at
3-hr. intervals. The hepatic cell nuclei were iso-
lated in citric acid, enumerated, lyophilized and
weighed. Desoxyribonucleic acid (DNA), ribo-
nucleic acid (RNA) and nucleoproteins were
measured by chemical methods and results were
expressed as mg X 10-*/nucleus. The average
weights of uninoculated control nuclei were 52.0,
from which the inoculated controls did not vary
materially throughout. No significant change
in weights of infected nuclei occurred during the
first 9 hr., but increased to 90.0 between 9 and 15
hr. Weights of nucleoproteins closely paralleled
weights of nuclei at corresponding intervals.
RNA did not change materially from 2.98 at 0 hr.
(uninoculated control) until after 9 hr. subsequent
to inoculation. Between 9 and 12 hr. the RNA
increased to 4.32 and remained at this level be-
tween 12 and 15 hr. DNA averaged 10.70 both in
control and infected cell nuclei. The finding of
increased weight of nuclei and of increased RNA
and nucleoproteins resulting from an animal virus
infection is unusual, if not unique. (Aided by a
grant from the Grayson Fndn.)

1652. Problems in standardization of isolated
myofibril contractile systems in _ vitro.
ARNOLD Brown,* ALBERT ARAS* AND GEORGE
Hass. Rush Lab. of Pathology, Presbyterian
Hospital, Chicago, Ill.

Previous studies showed that on milling cardiac
or skeletal muscle in a colloid mill, special condi-
tions were required before myofibrils could be
isolated in an uncontracted state and still retain
the ability to contract. Also, different prepara-
tions displayed different contractile reactions
which depended in part upon their age as well as
the concentration of ATP, Ca** or Mgt’. In
standardizing contractile systems of myofibrils
in vitro, it became necessary to inquire into these
differences. Canine cardiac and rabbit skeletal
muscle were milled in glycerol-buffer mixtures
(4:1) or buffer alone (0.154 m pH 7.0). The crude
homogenates consisting mostly of thick suspen-
sions of long myofibrils were studied. Fresh sus-
pensions failed to react to addition of ATP,

AMERICAN SOCIETY FOR EXPERIMENTAL PATHOLOGY

509

Cat+ or Mg** but when diluted with buffer (1:1
or more) the myofibrils promptly contracted from
long filaments down to dots. Similar suspensions
of myofibrils failed to contract on dilution, /)
if stored at 22°C for 2 hr., 2) if prepared from per-
fused muscle or muscle stored 24 hr. at 4°C, 8) if
washed or dialyzed. The contractile reaction on
dilution was restored to these ‘aged’ myofibril
suspensions by addition of ATP or the fresh
supernatant of an homogenate of fresh muscle.
These findings bear upon standardization of
in vitro systems for quantitative studies of proper-
ties of muscle by observing induction of con-
tractile reactions of myofibrils isolated from
normal or abnormal muscle when immersed in
supernatants from different muscle homogenates.

1653. Study of poliomyelitis virus by fluores-
cent antibody. Sonya M. BuckieEy* (intro-
duced by GritBertT DALLporF). Div. of Labs. and
Research, New York State Dept. of Health, Al-
bany.

The distribution of poliomyelitis viruses,
types I, II and III (Mahoney, MEF! and Leo
strains), was studied by means of fluorescein-
labeled antibody. Trypsinized monkey kidney
cells, grown in monolayers in tissue culture tubes
and retransplanted thereafter on cover glass,
were infected with large doses of virus. At various
postinoculation intervals, infected and nonin-
fected explants were overlaid with type-specific
human serums or human gamma globulin prior
to exposure to fluorescein-labeled antihuman
gamma globulin horse serum, pseudoglobulin
fraction. No specific fluorescence was observed in
uninoculated preparations or in inoculated
preparations harvested at 0 time. Thereafter
viral antigen was seen in the cytoplasm of infected
cells within 5 hr. after inoculation and within the
nucleus at later stages. The intensity of the cyto-
plasmic yellow-green fluorescence was greater
than that of the intranuclear. Antigen distribution
within the cytoplasm was diffuse or granular.

1654. Evaluation of intrinsic factor in perni-
cious anemia by means of cobalt 60-vitamin
Biz. Frances E. Buui,* Donatp C. CAMPBELL*
AND CHARLES A. OWEN, JR. Mayo Fndn. and
Mayo Clinic, Rochester, Minn.

Vitamin B,2 labeled with radiocobalt has been
used directly to evaluate the activity of intrinsic
factor and indirectly to diagnose pernicious
anemia. Three techniques have evolved for meas-
uring absorption of orally administered labeled
vitamin B,2: 1) fecal excretion (Heinle) ; 2). radio-
activity accumulated in the liver (Glass); 3)
urinary radioactivity after a ‘flushing’ dose of
unlabeled vitamin By: given parenterally (Schil-
ling). Ninety-two patients were studied by these 3

510 FEDERATION PROCEEDINGS

methods. A sharp distinction in absorption of
vitamin B;: was evident by any of these techniques
when normals were compared with patients having
pernicious anemia. The urinary method was sim-
plest and quickest, but unrecognized loss of part
of the specimen might suggest pernicious anemia
in a normal person. Renal excretion of radiocobalt
(Co®) in patients with uremia often was so re-
tarded that a single 24-hr. urinalysis gave mis-
leadingly low results; however, total excretion
over a period of several days was normal. The
fecal-excretion method was accurate but labori-
ous; unrecognized loss of specimens might yield
false normal values. Hepatic radioactivity was
measurable only after unabsorbed intestinal Co®
was excreted ; this method was slow but perhaps
most aceurate, since it is independent of the
patient’s cooperation in collecting excreta. When
initial tests indicated lack of intrinsic factor,
they were repeated with the addition of gastric
juice to the labeled vitamin; patients with per-
nicious anemia reverted to normal but patients
with severe sprue or intestinal disease often did
not.

1655. Relation of lymphoma induction to
cortical differentiation in thymic grafts in
irradiated C57BL mice. WiLL1AM H. CARNES
AND Henry S. Kaptan. Depts. of Pathology and
Radiology, Stanford Univ., San Francisco, Calif.
Systemic irradiation of C57BL mice induces a

high incidence of thymic lymphomas. Identical

tumors are induced in nonirradiated homologous
thymic grafts placed in previously thymecto-
mized, irradiated hosts (Proc. Am. Assoc. Cancer

Res. 2: 27, 1955). Irradiation of the host is essential

for the induction of tumors in the grafts. Initially

there is necrosis of all the implanted tissue ex-
cepting a narrow subcapsular zone. In nonirradi-
ated hosts thymic structure is regularly restored
by a proliferation of cells of this zone. Cortical
lymphocytes disappear from the grafts by the
7th day after implantation and are replaced by
newly differentiated lymphoid cells at the 10th-
14th day.-The earliest stages of thymic regenera-
tion in irradiated hosts are similar but the differ-
entiation of cortical cells fails to occur or is imper-
fect in a large majority of the grafts. Such grafts
are composed wholly or in large part of surviving
reticular or epithelioid cells resembling those of
thymic medulla. Shielding one thigh from the
radiation, which prevents the induction of lym-
phomas in the grafts, preserves the normal ca-
pacity for cortical regeneration. Thus a demon-
strated impairment of differentiation of cortical
lymphocytes is directly correlated with the induc-
tion of lymphomas in the thymus by irradiation.

(Supported by research grants from the Natl.

Cancer Inst., PHS, No. C778-5, and the American

Cancer Society, no. INSTR-78A.)

Volume 16

1656. Adrenal incorporation of C™“ from a
labeled bacterial polysaccharide: effect of
cortisone and ACTH. YoLaNnpDE CaRTER* AND
RussEL 8. Jones. Dept. of Pathology, Univ. of
Utah College of Medicine, Salt Lake City.

C from a Klebsiella pneumoniae polysaccharide
concentrates in the adrenals of the guinea pig and
persists for at least 2 months while the label
declines and disappears in the spleen and liver
(Proc. Soc. Exper. Biol. & Med. 90: 148, 1955). To
obtain some understanding of the factors involved
in the long persistence of label in adrenal, ACTH
and cortisone were given to separate groups of
guinea pigs. Daily injections of the 2 hormones
were begun 1 wk. before and simultaneously with
a single intravenous injection of polysaccharide
and continued until animals were killed 1 wk. after
polysaccharide injection. Cortisone was given in
2.5 and 10 mg doses, ACTH in 5 and 10 U.S.P.
units/250 gm animal. Tissue incorporation was
calculated as percentage of injected dose and as
percentage of tissue concentration (% gm/kg
b. wt.). ACTH led to a mild increase in adrenal
incorporation of label when calculated as _per-
centage of injected dose. However, the increased
adrenal weight resulted in slightly lower percent-
age of tissue incorporation. Cortisone, begun
before polysaccharide injection, causes a marked
decrease in incorporation of C™; compared to a
control value of 1.2% of injected dose, 2.5 mg of
cortisone depressed incorporation to 0.23% and 10
mg dose to 0.14%. Cortisone, begun simultane-
ously with the single injection of polysaccharide,
only mildly depressed C uptake. The tissue con-
centration of liver is mildly depressed with cor-
tisone apparently due to some increase in liver
weight; the percentage incorporation of injected
dose was not significantly altered.

1657. Energy -yielding reactions of Treponema
pallidum as studied by motility inhibition.
JaMEs D. Case AND J. WALTER CLARK, JR. (in-
troduced by Haroxtp J. Maanuson). Venereal
Disease Exptl. Lab., Chapel Hill, N.C.

The effect of various inhibitors on the motility
of 7’. pallidum has been studied in order to gain
insight into energy-yielding reactions of the
organism. Suspensions of motile organisms from
7-14 day rabbit testis syphilomas were incubated
at 35°C at several concentrations of inhibitor
under various atmospheres of oxygen, nitrogen
and carbon dioxide. The proportion of actively
motile organisms was determined by darkfield
microscopy on samples taken at 1-i hr. intervals
over a 24-30 hr. period. From this the time course
of decay of motility was plotted for control and
inhibitor suspensions. Inhibition of motility was
interpreted as indicating that the reactions in
question contributed to the energy required for

a a Se

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March 1956

motility’ work. Cyanide, azide, dinitrophenol,
fluoroacetate, malonate, sulfide, oxygen and
fluoride were used. Results indicated that oxida-
tive phosphorylation contributed little, if at all,
to motility energy. Preliminary experiments with
sulfhydryl inhibitors and fluoride suggested that
anaerobic energy-yielding reactions supplied the
bulk of motility energy.

1658. Reserpine suppression of murine
adrenal and reproductive responses to
population density. JouNn J. Curistian. Naval
Med. Research Inst., Bethesda, and Johns Hopkins
School of Hygiene and Public Health, Baltimore,
Md.

Adrenals of grouped mice increase in weight
over those of isolated controls due to cortical
hypertrophy; furthermore, the increase is propor-
tional to group size (Am. J. Physiol. 181: 477;
182: 292, 1955). Weights of secondary reproductive
organs were inversely proportional to group size.
These changes appeared to result from social
competition based on individual aggressiveness.
Moderate does of reserpine should prevent these
changes by suppressing aggressiveness (PLUMMER
et al. Ann. New York Acad. Sc. 59: 8, 1954).
Grouped and isolated male mice were given re-
serpine in water (approx. 507/mouse/day) and
compared with similar numbers of untreated
grouped and isolated mice. Treatment reduced
the number of fights between mice for the first
2 days after grouping. Fights in all groups were too
infrequent to measure after the second day.
Grouping produced an 8% increase in adrenal
weight in the untreated mice. Reserpine prevented
this increase, as an apparent 2% increase in
adrenal weight in the treated grouped mice was
not significant. The adrenal weights of the treated
and untreated isolated mice did not differ sig-
nificantly, nor did those of the isolated and
grouped treated mice. The weights of the seminal
vesicles and thymus glands varied inversely with
adrenal weight. The preputial glands weighed
significantly less in the treated than in the un-
treated grouped mice. There was no other sig-
nificant change in the preputial glands or testes.

1659. Inflammatory response to silica after
cessation of cortisone therapy. WALLACE
H. Criark, Jr., Letanp C. EpMONDS AND
Curton L. Hester (introuced by CHaR.LEs E.
Dunuap). Dept. of Pathology, Tulane Univ. Med.
School, New Orleans, La.

Three groups of male rats received, intra-
peritoneally, 50 mg of finely divided silica (av.
particle size 10-20 my) and 2 subcutaneous injec-
tions of 10 mg of coarse silica (av. particle size
2.9 wu). In group I the animals received no hor-
mones. In group IT and group III the animals

, YUM

AMERICAN SOCIETY FOR EXPERIMENTAL PATHOLOGY 511

received 5 mg of cortisone acetate daily, beginning
2 days before the injection of silica. In group II
the cortisone was continued throughout the
experiment and in group III the hormone was
stopped 4 days after silica injection. Animals
were killed in each group 1, 2, 4, 8 and 16 days
following cessation of cortisone in group III.
The liver and subcutaneous lesions were studied
microscopically. Four days after the injection of
fine silica in nontreated animals (group J), the
liver showed numerous ‘epithelioid’ cell tubercles.
The subcutaneous lesions in growp I showed an
early neutrophilic response followed by macro-
phages and fibrosis. In those animals receiving
cortisone throughout the experiment, no granu-
lomas developed in the liver and reaction to
subcutaneous silica was minimal. Two days follow-
ing cessation of cortisone (group III) the inflam-
matory response was apparent in the subcutaneous
lesions and liver. By 8 days the subcutaneous
lesions were similar to those in untreated animals
and many of the livers contained more granulomas
than the untreated controls.

1660. Spontaneous heart disease in DBA/2Jax
mice. GERALD R. CLEMENTS (introduced by
~W. B. Wartman). Dept. of Pharmacology, Ar-

mour Labs., Chicago, Ill.

A spontaneous disease which manifests itself
primarily in the muscle of the right ventricle of
the heart has been observed grossly at autopsy in
85% of DBA/2Jax mice. Clinically, some of the
mice show slight roughening of the coat and
lethargy. Many mice which appeared to be in good
health have shown right ventricular lesions of
varying extent at autopsy. A moderate functional
kyphosis is usually noted in mice which have
extensive cardiac lesions. Macroscopic examina-
tion reveals small, irregular, firm white plaques
on the surface of the right ventricle. In no case
has the muscle of the left ventricle been involved.
Occasionally small white flecks have been seen on
the surface of the liver and on section of the lobes;
these lesions occur only when the disease is ex-
tensive in the right ventricle. Lesions in the heart
and liver have been observed in mice 18 days old.
The right ventricular lesions are focal on micro-
scopic examination and consist of a mixture of
degenerated and necrotic myocardial fibers. Strik-
ing degrees of calcification are present in the
necrotic fibers and the degenerating muscle cells
stain positively with the PAS method. In animals
with extensive disease, Schiff-positive material
is frequently noted in the walls of the cardiac
vessels. Granulation tissue is seen in the areas of
extensive calcification. Infiltration of the cardiac
muscle adjacent to the lesions with inflammatory
cells is usually absent; occasionally a small ac-
cumulation of lymphocytes is seen.

512

1661. Occurrence of multiple fractures in
suckling rats injected with beta-amino-
propionitrile (Lathyrus factor). Jackson J.
CLEMMONS AND D. Murray ANGEVINE. Dept. of
Pathology, Univ. of Wisconsin Med. School.,
Madison.

Beta-aminopropionitrile (BAPN) has_ been
identified as the toxic ‘lathyrus factor’ in the sweet
pea, Lathyrus odoratus. Experiments with rats
have indicated that the type of bone lesion pro-
duced by the lathyrus factor varies with the age
of the animal. Young weanling rats develop
kyphoscoliosis and osteoporosis within 2-3 wk.
when fed the lathyrus-containing diet. Older rats
are less susceptible and do not develop deformi-
ties. To determine the effect of the lathyrus factor
on newborn animals, 3 rats from each of 3 litters
were injected subcutaneously at intervals with
0.1 ce of a 2% solution of BAPN and killed from
8 to 96 hr. later. A control rat was removed from
the same litter with each injected rat. Animals
from an additional litter received a larger amount
(0.3 cc) of BAPN and were killed within 12 hr.
The injected animals developed multiple fractures
of the long bones within 48-72 hr. after the initial
injection. x-rays of the animals at 72 hr. revealed
extensive osteoporosis. Histologic examination of
bones from animals killed 24 and 48 hr. following
injection with BAPN indicated that the fractures
were preceded by extensive degeneration of the
osteoblasts. Historadiographic studies of the bone
matrix revealed a decreased concentration of the
organic matter which is also associated with the
osteoporosis and degenerative changes in the
osteoblasts. There is some similarity between the
lesions produced by BAPN in baby rats and the
bone lesions observed in osteogenesis imperfecta.

1662. Conditionality and mammosomatotro-
pic features of estrogen-induced pituitary
tumors. Ke.iy H. Ciirron anp JAcoB FurTH.
Children’s Cancer Research Fndn., Boston, Mass.
Six estrogen-induced rat pituitary tumors have

been successfully transplanted in the strain of

origin. In‘the initial passage, none of the tumors
grew in normal females of this strain. All ‘took’
in estrogen-treated females. All hosts displayed
massive mammary gland stimulation with large
milk cysts. Tumor strain F4, which was most
extensively investigated, caused in addition,

a) marked enlargement of the liver with hyper-

trophy and hyperplasia of the parenchymal cells

similar to that found in mice with grafted mammo-
tropic tumors originally induced by radiation;

b) marked renal enlargement with proteinurea;

c) hypertrophy of the adrenal cortex with conges-

tion and extensive vacuolation of the cells. The

grafted tumor cells were nongranular or acido-
philic, and are thought to be nearly normal
mammotropes which proliferate when stimulated

FEDERATION PROCEEDINGS

Volume 18

by estrogen at about the same rate as did the mam-
motropes in the pituitary of the original host.
An autonomous variant of tumor strain 4 arose in
untreated females. Autonomous tumor cells
showed greater variation in nuclear and cell size
and morphology than the cells of dependent
tumors. Mammo- and somatotropic features were
still present and proteinurea was marked. Changes
induced in the mammary glands, livers and kid-
neys of estrogen-induced rat mammotropic tumor
hosts closely resemble those changes in mice
bearing grafts of radiation-induced mammotropic
tumors. The evidence suggests that the basic
induction mechanism of both estrogen- and radia-
tion-induced tumor types is a prolonged stimula-
tion of the pituitary mammotropes by estrogen.

1663. Effects of graded doses of whole-body
x-irradiation on mast cells in the rat mesen-
tery. FRANK P. ContrE,* Grorce 8. MELVILLE,
Jr.* anp ArtuuR C. Upton. Biology Div., Oak
Ridge Natl. Lab., Oak Ridge, Tenn.

In recent years, interest in the fate of mast cells
in irradiated animals has arisen in connection
with investigations of the hemorrhagic phase of
the radiation syndrome. A few investigators have
reported a decreased number of tissue mast cells
following lethal and supralethal doses of x-radia-
tion; however, others have observed an increase or
no change in the number of mast cells following
irradiation. The experiments to be reported were
designed to observe the mast cells in rats exposed
to graded lethal and sublethal doses of x-radia-
tion and to correlate changes in mast cell number
with dose and with time postirradiation. After 75r
of whole-body x-radiation, a transient increase
(20-30%) during the first 24 hr., followed by a
slight decrease, in the number of mast cells in the
rat mesentery was observed. Following a large
dose of radiation (600 r) the mast cells were seen
only to decrease in number during the first 2 wk.
after exposure, reaching maximally depressed
levels (40%) within 4 days after exposure to 600 r.

1664. Studies of lipid synthesis in cell-free
yeast extracts. LAURENCE M. Corwin, Law-
RENCE J. SCHROEDER AND WILLARD G. Mc-
CuLLoucH (introduced by ArtHur J. Vor-
wALD). Depts. of Physiological Chemistry and
Microbiology, Wayne Univ. College of Medicine,
Detroit, Mich.

A cell-free extract was obtained by shaking a
suspension of Red Star Bakers’ Yeast in 0.1 M
phosphate buffer at px 6.2 in a high speed bacterial
disintegrator (Corwin, L. M., L. J. ScHROEDER,
and W. G. McCuttouen. J. Am. Chem. Soc. 78:
1956. In press). The extract was dialyzed against
the 0.1 m phosphate buffer for 16 hr. A requirement
for additional cofactors in the conversion of 1-C™-
acetate to the fatty acids, sterols and squalene

stit
the

Grs
Na

166

,xXUM

me 16

mam-
host.
ose in
cells
1 size
ndent
| were
anges
1 kid-
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mice
tropic
basic
radia-
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zen.

-body
esen-
VILLE,
., Oak

t cells
ection
ase of
; have
t cells
radia-
ase Or
owing
| were
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radia-
umber
er 75r
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by a
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e seen
2 wk.
ressed
600 r.

l-free
Law-
. Mec-
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licine,

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-EDER,
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1-C¥-
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March 1956

could not be demonstrated. When the extract was
treated with Dowex 1-HCl, a requirement for CoA
was observed. In dialyzed extracts, however, it
was found that CoA was required for maximum
conversion of C'4-squalene to fatty acids. Cen-
trifugation of cell-free extracts at 11,500 g resulted
in the precipitation of yeast ‘mitochondria.’ In-
cubation of 1-C'4-acetate with the mitochondria
and with the supernatant resulted in a fairly equal
distribution of lipid-synthesizing activity. When
the extract was prepared in a saturated lactose
solution instead of buffer, the lipid-synthesizing
activity resided mainly in the mitochondria.
Microscopic examination of lactose and buffer
preparations of mitochondria stained with Janus
green showed clumping and partial disintegration
in buffer as opposed to individual, whole mito-
chondria in lactose.

1665. Histochemical changes in the rat kid-
ney induced by potassium deficiency. JOHN
M. Craig AND Rosert Scuwartz.* Children’s
Cancer Research Fndn., Depts. of Pathology and
Pediatrics, Children’s Med. Ctr. and Harvard
Med. School, Boston, Mass.

Twenty-nine young (120 gm) Sprague-Dawley
rats were placed for 14 days on a synthetic diet
which gave normal growth patterns when eaten
ad libitum, but which could be made deficient in
sodium and potassium by omission of either
sodium or potassium bicarbonate. This diet is
slightly modified from that used by Cohen,
Schwartz and Wallace (Arch. Path. 54: 119, 1952).
The animals were divided into 4 groups: 1) sodium
and potassium deficient, 2) potassium deficient,
8) sodium deficient, 4) sodium and potassium
supplemented diet, restricted to amount consumed
by group 2, §) sodium and potassium supplemented
diet fed ad libitum. The renal histological altera-
tions were essentially similar in the potassium
and sodium and potassium deficient animals,
though no similar changes were encountered in the
purely sodium deficient group. The changes in
the potassium and sodium-potassium deficient
groups included the well known dilatation of
Henle’s loops and the proliferation of cells in
certain tubules of the outer medulla. The potas-
sium deficient animals also displayed alteration
in their mitochondrial, PAS, PFAS, Smith-Die-
trich, Sudan black, Hale, esterase and acid phos-
phatase staining patterns in the ascending loops
of Henle, the collecting tubules and the inter-
stitium of the inner medullary zone. No change in
the alkaline phosphatase, or succinic dehy-
drogenase staining pattern was found. (Aided by
Grant C1975-C2 from the Natl. Cancer Inst.,
Natl. Insts. of Health, PHS).

1666. NaCl intake as related to human hyper-
tension. L. K. Daut anp R. A. Love.* Med.
Dept., Brookhaven Natl. Lab., Upton, N. Y.

E XUM

AMERICAN SOCIETY FOR EXPERIMENTAL PATHOLOGY

513

In 1954, evidence was presented which suggested
a relationship between NaCl intake and human
essential hypertension (DAHL AND Love. Federa-
tion Proc. 13: 426; Arch. Int. Med. 94: 525). Those
observations now have been extended to a total of
1346 consecutive adults with statistical analysis
indicating a high degree of significance to the
correlation between estimated salt intakes and
the presence or absence of hypertension. Subjects
were Brookhaven Laboratory personnel, seen
during annual physical examination by the
junior author, and salt intake classified as follows:
Low (Lo) had never used salt at table; Average
(Av) added salt to food at table if necessary but
only after tasting; High (Hi) always added salt
at table before tasting food. Among the 135, 630
and 581 members of Lo, Av and Hi there were 1,
43 and 61 hypertensives respectively (x? = 16.1,
P < .001). Although mean age was the same, sex
distribution differed with 25.2, 17.1 and 13.8%
females in Lo, Av and Hi. Analysis of the 1124
males was still significant (x? = 14.8, P < .001)
with the absence of hypertension in Lo signifi-
cantly lower (x? = 7.42, P < .01) and its presence
in Hi significantly higher (x? = 5.71, P < .02)
than predicted. The absence of salt craving in
normals or hypertensives after prolonged Na
limitation (4-8 mEq/d) implies that a high salt
intake is largely a taste habit rather than a symp-
tom of this disease. It is suggested that a high
salt intake, possibly over many years and par-
ticularly begninning in youth, is an important
etiological factor in the development of essential
hypertension.

1667. Growth of a plasmodial slime mold in
pure culture on a soluble medium. JoHN W.
DaniEL* anp H. P. Ruscu. McArdle Memorial
Lab., Univ. of Wisconsin Med. School, Madison.
Physarum polycephalum is a yellow-pigmented,

multinucleate ameboid myxomycete. A study of its

nutrition has been undertaken to facilitate the
study of factors controlling sporulation and of
the ensuing metabolic changes involved in the
transition from vegetation to sporulation. The
size of a single plasmodium, or ‘culture,’ depends
on the amount of substrate available, the range
commonly obtained being from 1 to 5 gm fresh
weight. Nuclear divisions in the plasmodial mass
are essentially synchronous. The organism may be
grown in still surface culture on a granulated agar
base saturated with liquid medium or on the

surface of liquid mediums as a continually mi-

grating syncytial mass. It produces an enveloping

layer of slime which is deposited as it migrates.

In shaken culture it grows as small but multi-

nucleate suspended plasmodia. Physarum has been

isolated in pure culture and grown at 20°C on 3

mediums: dry sterilized oats with added water; a

tryptone-glucose-yeast extract medium; an amino

514

acid-glucose-vitamins-salts medium. In the latter
2 mediums chick embryo extract is an absolute
requirement for growth without contributing
substantially as a nitrogen source. A number of
other natural materials are devoid of growth-
promoting activity. The growth optimum of pH 5
is maintained either by CaCO; or by phosphate
incorporated in the medium. Tryptone can serve
as a combined nitrogen and energy source but
growth is slower. A maximum yield of approxi-
mately 0.26 gm fresh wt/50 ml of medium is ob-
tained in 3} days with shaken culture and in 7-9
days with still surface culture when the medium
contains 1% tryptone and 1% glucose. Sustained
growth of this organism in pure culture on a
soluble medium has not been previously reported.

1668. Identity of renal lesion in rats treated
with antikidney serum. W. E. Exuricns, G. P.
SHarma,* R. A. Rasou,* N. BHAMARAPRAVATI*
AND J. SEIFTER. Univ. of Pennsylvania, Grad.
School of Medicine, Philadelphia.

Some 180 rats weighing 50-100 gm were injected
intravenously with 0.025-0.8 ml/100 gm of rat
weight of the globulin fraction suspended in
saline of the same anti-rat-kidney serum; 0.2 ml
of this suspension was fatal to 28%, 0.4 ml to 67%
and 0.8 ml to 89% of the animals within 1 wk. after
injection. The surviving rats were killed on the
3rd, 7th, 14th and 30th day of the experiment.
Many rats injected with 0.025-0.075 ml of the
globulin suspension developed diffuse endothelial
proliferation of all glomeruli indistinguishable
from the glomerular lesion of acute diffuse glo-
merular nephritis in man (progressive glomerulo-
nephritis). The urine contained a moderate con-
centration of protein. No edema nor ascites was
found. Most of the rats which received 0.1-0.8
ml of globulin suspension, on the other hand,
showed the picture of lipid nephrosis, that is
swelling of the filtering membrane of all glomeruli
with or without thrombosis and _necrobiosis
(regressive glomerulonephrosis). The urine con-
tained large quantities of protein and most of the
rats revealed ascites at autopsy. Mixed forms
betweén-the nephritis and nephrosis also occurred.
These observations seem to show that glomerulo-
nephritis and lipid nephrosis may be etiologically
the same, but they differ pathogenetically in that
nephritis is a progressive and nephrosis a regres-
sive disorder. Also, lipid nephrosis is the result of
a more serious injury.

1669. Growth of monkey kidney cells in dif-
fusion chambers in mice; effect of polio-
myelitis virus. ALLEN B. EscHENBRENNER AND
Rosert D. Francis.* PHS, Communicable Dis-
ease Ctr., Montgomery, Ala.

Trypsin dispersed monkey kidney cells sus-

pended in a nutrient medium, consisting of 199

FEDERATION PROCEEDINGS

Volume if

mixture, horse serum and Hanks’ solution, we
grown in vivo in diffusion chambers implantej
subcutaneously in mice. Rectangular chamb
frames with centered holes were fabricated fron
Plexiglas. A cellulose derivative membrane filte
having an average pore diameter of 1 uw was ¢
mented on one side of the frame. The resulting
well was filled with cell suspension and a coversliy
was then cemented on top, thus forming a sealej
chamber having a glass side on which cells coul
grow and a porous side for interchange of metabe
lites. The chambers were implanted sub
cutaneously in mice with the membrane side @
the chamber adjacent to the abdominal muse
ture. Seven to 10 days later tissue culture fluid
containing approximately 10,000 Tc1Ds of type
(Mahoney strain) poliomyelitis virus was inoey
lated into the potential space existing between th
membrane side of the chamber and the abdomirz
musculature. Controls were inoculated with vi
free tissue culture fluid. One, 2 and 3 days afte
inoculation, chambers were removed and micr
scopic observations were made of the condition
the cells adhering to the coverslips. The latter the
were removed, stained and mounted on micnm§ .
scope slides. The morphological and _tinctorial
changes in the cells growing in vivo following
inoculation of the virus were similar to tho
observed in vitro.

1670. Effect of hypothermia upon induce
bacteremia. E. J. Fepor,* E. R. Fisuer, 8
FisHER AND J. V. Dattiio.* Depts. of Pathology

and Surgical Research, Univ. of Pittsburgh

Pittsburgh, Pa.

During the course of investigations concerning
the effect of hypothermia and rewarming upon the
sterility of the blood, it was observed that an-
aerobic and aerobic blood cultures remained sterile
in 12 adult dogs subjected to this state. Thes
results indicated that the gastrointestinal trae
and other bacterial foci maintained their in
tegrity throughout hypothermia and rewarming
To ascertain the effect of cooling upon induced
bacteremia 5-50 billion E. coli and A. aerogenes
were intravenously injected into 5 normothermit
and 12 hypothermic dogs. The bacteria were ob-
tained from stool cultures of the animals utilized
in the experiments. Blood cultures of the normo
thermic dogs were all sterile within 24 hr. following
injection, whereas positive blood cultures @
these organisms were obtained in all but one @
the hypothermic dogs at this period. However, al
animals revealed clearance of bacteria from thé
blood within 24 hr. after rewarming. Morbidity
mortality was not observed in the hypothermit
dogs suffering from bacteremia. The results @
this study indicate that although hypothermis
does not provoke the state of bacteremia th
mechanisms involved in the clearance of bacteri

Volume if

tion, wer
implante;
- chamber
ated fron
rane filte
WAS ee.
> resulting
4 coversliy
ig a seale/
cells could
of metabe
ted — sub
ine side 9
| muse
lture fluid
of type
vas inoct
tween the
abdomin
vith vi
days afte
nd micro
ndition @
atter the
on micro
tinctorial
following
to thos

induced
ISHER, B;
Pathology)
-ittsburgh

oncernily
‘upon the
that an-
1ed sterile
te. Thes
inal tract
their in
warming,
1 induced
aerogenss
10thermit
were ob
s utilized
e norm
following
Itures of
ut one
wever, all
from the
rbidity 0
yothermit
esults @
othermis
emia th
' bacterii

March 1956

from the blood are depressed during this state.
(Supported’ in part by Public Health Service
Grant no. 2065.)

1671. Effect of estrogen on thyroidal and renal
clearance of I'*!, JosepH D. Fretpman. Dept.
of Pathology, Univ. of Pittsburgh School of Medi-
cine, Pittsburgh, Pa.

Estrogen was administered to castrate male and
hypophysectomized female rats for brief and pro-
longed periods. Thyroidal clearance of I'*! was
studied over a 30-min. and a 24-hr. period; renal
clearance of I'#! was studied over a 24-hr. period.
Thyroidal clearance of I'*! was significantly in-
creased in animals treated with estrogen for 3-5
days; thyroidal clearance was unaltered in animals
treated for 15-35 days with estrogen compared to
control rats. Renal clearance of I'*! was slightly
depressed (not significantly) in rats treated with
estrogen for 3-5 days, as compared to renal clear-
ance in controls. The distribution of I'*! in estro-
gen-treated and control animals was the same.
The plasma concentration of I'*! was always
greater in estrogen-treated rats, averaging 15%,
but insufficiently elevated to cause a 2-fold
increase of thyroidal clearance of I'*!, The data
indicate a direct action of estrogen on thyroid
physiology without the mediation of the pituitary.
(Supported by Public Health Service Grant no.
C-2579.)

1672. Regulation of cytoplasmic ribonucleic
acid and protein synthesis in endocrine
glands by hormones of anterior pituitary.
Strvio Fraua,* Epirx E. Sprout anp ANNA
Frata.* Dept. of Pathology, Columbia Univ.
College of Physicians and Surgeons; Francis
Delafield Hosp., New York City.

The tropic hormones of the anterior pituitary
gland such as thyrotropin (TSH), folliculin (FSH),
corticotropin (ACTH) and prolactin induce cyto-
plasmic growth in their target organs without
inducing to any greater degree cellular divisions.
For example, a 40% increase in the weight of rat
thyroid induced by 5 injections of purified TSH
(Armour) in the course of 48 hr. is related to an
80% increase of cytoplasmic RNA with a cor-
responding increase of cytoplasmic proteins,
while DNA remains practically constant. Simi-
larly, an approximately 25% increase in the
ovarian cytoplasmic RNA occurs within 12 hr.
after a single administration of 2 mg of purified
FSH (Armour) without any detectable increase in
DNA in young hypophysectomized female rats.
ACTH has a similar effect on the adrenal (J.
Biochem. Biophys. Cyt. 1956. In press) and the
same is true for prolactin. An induction period of
3-5 hr. precedes the detectable increase in RNA,
while a deep drop in acid-soluble substances
absorbing at 260 my can be detected within 1-2

AMERICAN SOCIETY FOR EXPERIMENTAL PATHOLOGY

515

hr. after administration of RSH, FSH or ACTH.
The growth of the cytoplasm in all these cases is
related to an increase of the basophilic quotient
(RNA/protein) of the sedimentable chromidial
fraction.

1673. Dual mechanism of pathogenesis in in-
fections with cyst-producing microorgan-
isms. J. K. FRENKEL. Dept. of Pathology and
Oncology, Univ. of Kansas School of Medicine,
Kansas City.

Concepts of pathogenesis concerning the
prevalent infections hold considerable generaliza-
tion value. Infectious agents proliferate in a host
and exert their effects until immunity supervenes,
after which lesions cease to appear. In certain
infections following a chronic course, effective
immunity is established slowly, often after several
remissions and relapses. In both acute and chronic
infections, lesions are associated with proliferation
of organisms. An additional mechanism of disease
production exists in infections with certain cyst-
producing organisms. Intact Toxoplasma cysts in
brain, eye, skeletal and heart muscle offer no
evident chemotactic stimuli. Rupture of cysts
leads to an acute necrotizing reaction, suggesting
hypersensitivity, and to destruction of the liber-
ated organisms. There is no active proliferation of
Toxoplasma. Depending on the number of cysts
and frequency of their rupture, lesions of func-
tional significance to their host may result, such
as encephalitis, retinitis, myocarditis and myosi-
tis. A certain proportion of cases of retinochoroidi-
tis in man are probably caused by rupture of
Toxoplasma cysts. Cysts are too small to be de-
tectable by ophthalmoscopy and remnants of
cyst wall are rarely observed in sections. Cysts
can be visualized ophthalmoscopically in hamsters
infected with a larger protozoan, Besnoitia jelli-
soni (Am. J. Ophth. 39: 203) and lesions resulting
from cyst rupture in the eye and other organs are
marked histologically by the persistence of the
heavy cyst wall. Hence, in induced and spon-
taneous infections with Toxoplasma and Besnoitia,
lesions can be produced by proliferation of or-
ganisms, as well as by the sudden release of
antigen incident to the rupture of cysts.

1674. Differential effects on cellular division
and differentiation. NATHAN B. FRIEDMAN,
Rosert E. Hoyt* anp E1LeEN Drutz.* Div. of
Labs., Cedars of Lebanon Hosp. and Dept. of
Pathology, Univ. of Southern California, Los
Angeles.

Previous work has shown that radiation and
certain chemotherapeutic agents do not interfere
with differentiation of intestinal epithelium
although they do inhibit division. Other chemical
agents affect both division and differentiation.
These studies have been extended to include other

516

biological systems. Certain effects in transplant-
able tumors and bacterial cultures suggest that
the dichotomy between division and differentia-
tion may represent a general phenomenon. (Sup-
ported by Public Health Service Grant C2490.)

1675. Metabolism of hydrocortisone in iso-
lated perfused dog kidney. Franx M. Gants
(introduced by J. J. Morton). Depts. of Radia-
tion Biology and Biochemistry, Univ. of Rochester
School of Medicine and Dentistry, Rochester, N.Y.
A kidney was removed from a male dog and

perfused at 38°C with oxygenated homologous
blood containing hydrocortisone. The perfusate
was extracted with chloroform and the latter
reduced in volume and treated with methanol and
petroleum ether to remove nonsteroidal lipides.
The steroid-containing residue was then analyzed
by paper chromatography using techniques
previously described in detail. (Ganis, AXELROD
AND MiutEr. J. Biol. Chem. In press). Twenty-one
discrete fractions showing qualitative reactions
characteristic of steroids were isolated. Of these,
4 were identified by various spot tests, by mixed
chromatograms with authentic compounds, by
absorption maxima in methanol and concentrated
sulfuric acid and by the preparation of suitable
derivatives of those compounds present in suffi-
cient amount. The identified compounds are:
A‘-pregnene-118,17a,208,21-tetrol-3-one, A‘-preg-
nene-17a,21-diol-3,11,20-trione, A‘-androstene-
118-0l-3,17-dione and A‘-androstene-3,11,17-
trione. At least 4 of the fractions isolated retained
the A‘-3 ketone grouping of ring A. The metabolite
recovered in greatest quantity was the C-20 re-
duction product of hydrocortisone. As indicated
above, metabolically active androgens were pro-
duced by oxidative cleavage of the side chain.
These may play a role in the course of metastatic
carcinoma of the prostate in orchiectomized and
adrenalectomized humans maintained on adreno-
cortical hormones. These findings on dog kidney
are in agreement with earlier observations on
hydrocortisone metabolism in bovine kidneys.

1676. Electron microscope study of experi-
mental “necrotizing arteritis in the dog.
Jack C. Grrr,* Henry C. McGiIu, Jr. anp
RusseEuu L. Hotman. Dept. of Pathology, Louisi-
ana State Univ. School of Medicine, and the
Electron Microscope Lab., Charity Hosp. of
Louisiana, New Orleans.

Renal insufficiency in the dog is known to result
in necrotizing lesions of several components of
the vascular system. This study has been pri-
marily concerned with the pathogenesis, as dis-
closed by the electron microscope, of necrotizing
lesions in the walls of the great vessels and the
auricular endocardium which are produced only
when dogs have been prefed a high fat diet rich

FEDERATION PROCEEDINGS

Volume i

in creamery butter. These lesions resemble closely
the lesions of certain of the ‘collagen diseases’ seen
in humans. Under the electron microscope, the
earliest change in the vessel wall preceding a
definite necrotizing lesion is an increase in finely
reticular interstitial material accompanied by
leucocytic infiltration. Later platelets and de.
posits of fibrin appear and the interstitial inter.
fibrillary material becomes homogeneous. Only in
well-advanced lesions are there degenerative
changes in the smooth muscle and elastic tissue
and the unit fibers of collagen persist unaltered
even longer. The lesions mentioned above de-
pendent on prefeeding the butter-rich diet are
to be contrasted with the necrotizing arteritis and
arteriolitis produced by renal insufficiency alone,
Among other differences, the electron microscope
indicates that this latter lesion consists primarily
of necrosis of smooth muscle, rather than first
affecting the interstitial substance.

1677. Effect of aminopterin and omega-
methyl-pantothenate combined on mor-
phology and _ respiration of intestinal
mucosa. THomas J. Giuu, III,* Norman Zam-
CHECK, JOSEPH J. VITALE* AND D. M. Hegstep.
Dept. of Nutrition, Harvard School of Public
Health, the Mallory Inst. of Pathology and Dept.
of Pathology, Boston Univ. School of Medicine,
Boston, Mass.

Rat intestinal mucosa is susceptible meta-
bolically and morphologically to deficiencies of
pantothenic acid and folic acid (aminopterin).
Aminopterin (240 ug/kg b. wt.) caused death in
20-21 days with typical findings of lethargy,
weight loss, alopecia, bloody diarrhea and with
characteristic morphologic changes (J. Lab. &
Clin. Med. 43: 583, 1954). Omega-methyl-panto-
thenate (750 ug/kg b. wt.) had no clinical or
morphologic effect. When combined with aminop-
terin in the same dosage, however, the animals
died in 6-12 days. Microscopic degeneration and
inflammation of the ileal and colonic epithelium
was observed. In in vitro studies the combined
addition of aminopterin (120 ug/Warburgh flask)
and omega-methyl-pantothenate (1500 ug) caused
a marked depression in respiration from a control
QO: of 15.7 to 3.9. Neither of these analogues alone
in comparable doses depressed the respiration
significantly. The enhanced effect of combined
aminopterin and omega-methyl-pantothenate on
metabolism and morphology suggests a possible
synergistic action of pantothenic acid and folie
acid on the gastrointestinal mucosa.

1678. Silicosis; topographic relation of silica
to pulmonary tissue. Paut Gross, Marian L.
Westrick* anp JAMES M. McNerney.* [ndus-
trial Hygiene Fndn., Mellon Inst., Pittsburgh, Pa.
A newly developed method for demonstrating

lume 16

: closely
es’ seen
ype, the
eding a
n finely
tied by
and de-
ul inter-
Only in
nerative
c tissue
naltered
ove de-
liet are
‘itis and
y alone,
TOScope
rimarily
an first

omega:
2 mor-
estinal
.N ZaAM-
EGSTED,

Public
ul Dept.
edicine,

. meta-
ncies of
pterin).
leath in
thargy,
id with
Lab. &
|-panto-
tical or
uminop-
animals
ion and
thelium
mbined
h flask)
caused
control
es alone
piration
mbined
nate on
possible
nd folic

f silica
RIAN L.

March 1956

mineral particles in their topographic relationship
to histologic structures consists of taking photo-
micrographs of carefully located fields in a stained
section and after incinerating the section, treating
with acid and washing, rephotographing the
same fields but under dark-field conditions. The
negatives thus obtained are carefully superim-
posed, matched and bound together with trans-
parent tape. From these, composite prints are
obtained. During a study of over 300 photomicro-
graphs it has been observed that the mineral
content of silicotic rat, and human, lungs is
variable from one nodule to the next. The vari-
ability of the mineral content is considered to be
caused by a demineralization process which, in
turn, is mediated by edema from whatever cause.
Mineral flocs are frequently found within rela-
tively uninvolved tissue or the air spaces adjoin-
ing demineralized nodules. It is concluded that
the movement of silica from well established
nodules to uninvolved lung tissue and to air spaces
adequately explains the recognized progressive-
ness of silicosis and the unreliability of the silica
content of lung tissue as a criterion for the diag-
nosis of silicosis.

1679. Mode of action of insulin as related to
lipogenesis in isolated perfused rat liver.
Davin E. Hart (introduced by Eric L. ALLIN@).
Atomic Energy Project, Univ. of Rochester School
of Medicine and Dentistry, Rochester, N. Y.

The role of insulin in intermediary metabolism
of the isolated rat liver was investigated through
use of a perfusion technique (MILLER et al. J.
Exper. Med. 44: 431, 1951) in which acetate-1-C™
and varying concentrations of unlabeled glucose,
fructose or lactate were added to the circulating
blood. Livers removed from alloxan-diabetic
rats responded to the direct addition of insulin
by correction of the decreased lipogenesis and
increased gluconeogenesis characteristic of dia-
betes. Without added insulin, high concentrations
of glucose and fructose caused a net disappear-
ance of substantial quantities of carbohydrate in
diabetic and normal livers but failed to simulate
the insulin effect on incorporation of acetate into
fatty acids. Unlabeled lactate, on the other hand,
was as effective as insulin in stimulating such
incorporation. These observations are consistent
with the view that insulin affects lipogenesis by
increasing the ratio [DPNH]/[DPN], but that this
change in ratio does not result merely from an
increase in sugar uptake.

1680. Growth and serial transfer of a mouse
leukemia in cheek pouch of the golden
hamster. ALFRED H. HANDLER (introduced by
StpneY Farser). Children’s Cancer Research
Fndn., Boston, Mass.

This investigation was undertaken in an at-

AMERICAN SOCIETY FOR EXPERIMENTAL PATHOLOGY

517

tempt to grow leukemic cells from an alien species
in the cheek pouch of the golden hamster. Lym-
phatic leukemia, P1534, which is 100% transplant-
able in DBA-2 mice and which is maintained in
our mice as a solid tumor, implanted intramuscu-
larly, was implanted by trocar into the cheek
pouches of 10 young adult golden hamsters. Five
of the hamsters received subcutaneous injections
of cortisone acetate (3 mg twice/week) and 5 re-
mained untreated. Tumor growth was observed in
all of the hamsters treated with cortisone acetate
and in 3 of the 5 untreated animals. Tumors in the
cortisone-treated animals increased in volume
almost 2000 times in 10 days, while in untreated
animals tumor growth was somewhat slower.
P1534 has been perpetuated by passage from
hamster to hamster and is at present in the 14th
transplant generation. From the 6th through the
14th passage, P1534 grew in all of 45 untreated
hamsters and in only 27 of 45 hamsters treated
with cortisone acetate. The tumors in untreated
hamsters have grown more rapidly after the 5th
passage and at present fill the cheek pouches in
10 days, while the tumors in cortisone-treated
animals grow at a slower rate. Although P1534
grows vigorously in hamsters, it invades only the
tissues adjacent to the cheek pouch and does not
demonstrate generalized leukemia. Twelve days
after implantation tumors ulcerate the cheek
pouches and become necrotic. Hamsters usually
die of secondary infection after 30-60 days. Micro-
scopically the tumors appear as lymphomas. After
the 9th, 12th and 14th passages in hamsters,
P1534 was reimplanted successfully into 100
DBA-2 mice by intramuscular, intravenous and
intraperitoneal routes. The mice died after 11-16
days, many with generalized leukemia. P1534 has
failed to grow progressively in LAF or BAF mice,
in rats or in Chinese hamsters. (This investigation
was supported by a research grant, C-2230 (C),
from the Natl. Insts. of Health, PHS.)

1681. Osmotic control of cytochrome oxidase
and succinic dehydrogenase activity in mus-
cle mitochondria. JoHn W. Harman. Dept. of
Pathology, St. Kevin’s Hosps., Dublin, Ireland.
Mitochondria of cardiac and pigeon breast

muscle are isolated in 1.0 m sucrose. Resuspen-

sion in osmotic gradients of sucrose between 1.0

and 0.05 m sucrose establish a gradual transition

of rodlets into spheres and vesicles (crescents)
which statistically reflects the osmolar level of
the medium. Cytochrome oxidase activity meas-
ured by the indophenol-blue technique of Straus
and succinic dehydrogenase activity determined
by the formation and spectrophotometric estima-
tion of the formazan from neotetrazolium are
similarly affected by osmolarity. Both enzymes
parallel the morphological transformations,
being minimal at 1.0 m sucrose and maximal at

518

0.05 m sucrose where swelling is greatest. Disrup-
tion of the mitochondria mechanically or by
freeze/thawing eliminates osmotic reactivity
and establishes maximal enzyme activity in the
subparticles. Both morphology and enzyme
activity are partly reversed by resuspension in
higher osmolar mediums; particles are not so
affected. After washing with 0.5 M KCl, the cyto
chrome c is eluted; with readdition of cytochrome
c the osmotic gradient response in sucrose is still
manifest. It is apparent that intact mitochondria
are necessary for osmotically induced alterations
in both morphology and activity of individual
enzyme of the electron chain. Although the
integrated succinoxidase is repressed by fall in
the osmolarity, the activities of component
enzymes are augmented. Since activity of mito-
chondrial-bound enzymes is affected by osmolar
and structural alterations careful control of such
factors is required in comparative assays.

1682. Procedure for sterilization of plasma
using combinations of ultraviolet irradia-
tion and beta-propiolactone. FRANK W.
Hartman, GERALD A. LoGrippo* anp ANNETTE
R. Keuty.* Dept. of Labs., Henry Ford Hosp.,
Detroit, Mich.

It was shown in previous communications that
with a battery of different viruses the titer could
be readily reduced to the point where cultures
were negative with concentrations of 2000-3000
mg of beta-propiolactone/l. of seeded plasma.
However, animal inoculations of this treated
plasma resulted in a small percentage of infections
due to trace quantities of uninactivated virus
(tailing effect). This ‘tailing effect’ was eliminated
by doubling the dosage of beta-propiolactone 4000
to 6000 mg/]. of plasma but this dosage produced
changes in the plasma proteins. Apparently a
similar ‘tailing effect’ occurs with the optimum
dosage of ultraviolet radiation to plasma result-
ing in 4-7% failure to prevent hepatitis after the
administration of the treated plasma. The use of
beta-propiolactone in combination with ultra-
violet irradiation produced complete inactivation
of 10% virus suspensions of EEE and MM viruses
when seeded in 90% human plasma. Our tests
indicate that drug concentrations of } to § and
ultraviolet irradiation of } to } in combination
equal the inactivation achieved with either beta-
propiolactone or ultraviolet irradiation alone. The
procedure includes adjustment of the pu of the
plasma and chilling before the addition of the
beta-propiolactone with constant vigorous shak-
ing in a closed bottle during the addition. After
the plasma has had beta-propiolactone added, it
is passed through the Dill ultraviolet irradiation
apparatus into a second Pyrex bottle. This bottle
is in turn gradually warmed to 37°C with constant
shaking and adjustment of the px to 6.8 through

FEDERATION PROCEEDINGS

Volume 15

the addition of NaOH to neutralize the acidity
produced by the hydrolysis of the beta-propiolac-
tone.

1683. Periportal stainable lipid in livers of
insulin-injected rats. W. STANLEY Hartrort
AND JOHN 8. Meyer.* Dept. of Pathology, Wash-
ington Univ. Med. School, St. Louis, Mo.
Groups of 30 male Wistar rats (100 gm) were

fed ad libitum a basal hypolipotropic diet supple-

mented with either 0.05 or 0.5% choline chloride.

Twenty of each group were subcutaneously

injected b.i.d. with increasing amounts of insulin

up to 16 u/day for periods up to 8 wk.; the re-
mainder received equal volumes of saline. Food
intakes and body weights of insulin-treated rats
exceeded those of saline controls. At autopsy,
granules were restricted to vascular poles of
pancreatic beta cells in the insulin-treated ani-
mals. In a significant number of their livers,
periportal stainable fat was present but was absent
in those of saline controls; abnormal fat in this
region (periportal) had not been influenced by

the level of dietary choline. But choline (0.5%)

had prevented accumulation of centrolobular fat

(present in livers of rats receiving only 0.05%

choline) which was, however, unaffected by in-

sulin administration. The treatment accorded
each rat was therefore manifested in sections of

most of their livers as shown in table 1.

TABLE 1

| 0.05% Choline | 0.5% Choline

Insulin | Saline

| ee)

Centrolobular fat
Periportal fat

+/+} 0 | 0
+ | 0] +] 0

These results support the concept that fat deposi-
tion in periportal lobular positions is controlled
by one set of factors and that in centrolobular
sites by others. (Supported by grants from the
Lipotropic Fndn. and the Public Health Service,
C2548.)

1684. Production of endocarditis and glo-
merulonephritis by single bacterial injec-
tions in dogs with aortic insufficiency.
BENJAMIN HIGHMAN, JOSEPH RosHE* AND PAUL
D. AutrLanp. Natl. Inst. of Arthritis and Meta-
bolic Diseases and Clinic of Surgery, Natl. Heart
Inst., Natl. Insts. of Health, Bethesda, Md.

As reported previously (Am. J. Physiol. Dee.
1955), endocarditis is readily induced in dogs with
aortic insufficiency by multiple intravenous
bacterial injections. The present study was under-
taken to determine the effect of a single bacterial
injection. Aortic insufficiency was induced in 6
dogs by fenestrating the right or left coronary

tan GP pe Pin ee ee oe me et tt ck mk

me 16

cidity
yiolac-

‘rs of
TROFT
Wash-

) were
upple-
loride.
eously
nsulin
she re-
. Food
d rats
itopsy,
les of
d ani-
livers,
absent
in this

ions of

Choline

Saline

| 0
| 9

deposi-
itrolled
lobular
om the
Service,

d_ glo-
injec-
ciency.
‘iD PAUL
d Meta-
l. Heart
‘
ol. Dee.
»gs with
avenous
s under-
acterial
ed in 6
oronary

March 1956

cusp (Rosie AND Morrow. Surg., Gynec. & Obst.
101: 305, 1955). The femoral arterial pulse pres-
sures ranged from 100 to 150 mm Hg. One cubic
centimeter of a 5-hr. bacterial broth culture was
injected intravenously 10-21 days after surgery.
Three dogs received Staphylococcus aureus and
were killed preterminally in 3-6 days. Autopsies
revealed multiple infarcts and an acute endo-
carditis involving the aortic and mitral leaflets;
hemorrhagic and suppurative lesions were found
in nearly all organs. The 3 other dogs received
Streptococcus mitis JH26 (Lancefield D) and were
killed in 10-14 days. All showed bacterial vegeta-
tions involving the aortic and mitral valves.
There were renal and splenic infarcts and scattered
small hemorrhagic and inflammatory lesions in
various organs; 2 of the 3 dogs showed a diffuse
proliferative glomerulonephritis. The lesions were
similar to those found after multiple bacterial
injections. This single-injection method facilitates
the study of experimental endocarditis and glo-
merulonephritis. It will be useful in testing in vivo
the efficacy of various forms of therapy.

1685. Lipid concentration of human adrenals
with particular reference to hypertensive
disease. CorRNELIA Hocu-Licretr1, KAREN Ir-
VINE* AND JAMES E, IrvinE.* Dept. of Pathology,
Univ. of Virginia, Charlottesville.

The adrenals from 230 unselected autopsies
were analyzed. for cholesterol, phospholipid and
total lipid concentration; the heart, kidney, liver
and blood of 40 of these cases were similarly
studied. The data were correlated with the disease,
cause of death, degree of arteriosclerosis, the
weights of the adrenals, kidneys and heart. Based
on data in the literature and on our findings in
cases of accidental sudden death, the normal
adrenal cholesterol concentration may be con-
sidered to be 4-6 gm/100 gm wet tissue. The
average concentration of cholesterol, total lipids
and dry weights of adrenals were significantly
increased in groups of patients dying with com-
pensated hypertensive cardiovascular disease.
In individual cases values up to 3-4 times normal
were found. The adrenal cholesterol concentra-
tion was significantly decreased in patients with
malignant diseases and acute infections; in those
with chronic infections or degenerative diseases
it was slightly decreased. In persons who com-
mitted suicide the adrenal cholesterol concentra-
tions were significantly increased. The increase of
cholesterol concentration in hypertension was
restricted to the adrenals; that of the heart, kid-
ney, liver and blood did not differ from the normal.
The phospholipid concentrations in the organs
did not differ significantly in the diseases studied.
On histological investigation the adrenals in
hypertension showed increase of lipid content in
the zona fasciculata and of brown pigment con-

. XUM

AMERICAN SOCIETY FOR EXPERIMENTAL PATHOLOGY

519

taining cells in the reticularis, apparently charac-
teristic for that disease.

1686. Successful cross-species bone grafting
accomplished by removal of the donor or-
ganic matrix. LLtoyp A. HuRLEY AND F Rep L.
LosgE (introduced by E. W. Gooppasture).
Naval Med. Research Inst., Natl. Naval Med.
Ctr., Bethesda, Md.

Cortical bone from rat, beef and man was
treated to remove organic components and
grafted into the tibias of normal adult dogs. The
organic matrix was removed by constant boiling
of ethylenediamine in a Soxhlet extraction appara-
tus for 48 hr. followed by absolute alcohol for 4
hr. The resulting bone presented an essentially
sterile, very white, easily shaped and extremely
porous inorganic matrix. The bone was placed in
experimental cortical defects 20 x 5 mm by either
inlay or chip procedure. Weekly roentgenograms
were taken and weekly intraperitoneal injections
of alizarin red S were given to record reparative
action. Six dogs were grafted with rat bone, 5 dogs
with bovine bone and 6 dogs with human bone.
Evaluation was made by the usual histological
methods supplemented by the use of micro-
radiography, fluorescent photomicrography,
polarized light and time lapse cinemicropho-
tography of the demineralization of 100-z sections
in a chelating agent. Acceptance was ascertained
by the revascularization, mineral bonding of the
donor to host bone, absorption and remodeling
of the inorganic implant, and by perivascular new
bone formation.

1687. Interferometric dry mass determina-
tion of isolated liver nuclei from fasting and
fed mice. Susumu Io (introduced by CrctLie
LEUCHTENBERGER). Inst. of Pathology, Western
Reserve Univ., Cleveland, Ohio.

In the present study dry mass of isolated liver
nuclei from fed, fasted and refed mice was deter-
mined by interference microscopy. The liver
nuclei were also examined for tyrosine and DNA
content (microspectrophotometry of Millon and
Feulgen reactions). The liver nuclei were isolated
in 10% formalin and in citric acid. Nuclei isolated
in formalin gave almost twice the dry mass and
tyrosine values of nuclei isolated in citric acid.
The DNA values of formalin-isolated nuclei were
the same or slightly higher than those after citric-
acid isolation. The mean dry mass (in 10-! gm)
for diploid nuclei isolated in formalin ranged
between 36 and 43 in animals fasted for 3-44 days.
Control mice had values between 40 and 55 while
nuclei from fasted and subsequently fed mice
ranged in mass from 42 to 58. The lowest liver
nuclear mass was found in animals which had
lost the greatest amount of weight by prolonged
fasting. Starved animals which were refed did not

520

show increased nuclear mass up to 6 hr. after
feeding but a marked increase after 12 hr. The
tyrosine values showed the same relationship
as the dry mass values. Polyploid nuclei had mass
and tyrosine values in approximate multiples of
the diploid values. DNA values showed no differ-
ence in the nuclei between fed, fasted and refed
mice.

1688. Hepatic changes following ligation of
common bile duct in the rat. WILLIAM E.
Jaques aND A. JAMES McApams.* Depis. of
Pathology, Louisiana State Univ. School of Medi-
cine, New Orleans, and Harvard Med. School,
Boston, Mass.

Sixty-nine white rats were used in this experi-
ment and were divided into 3 groups. The Ist
group of 2 rats served as litter mate controls. The
2nd group of 28 rats had a complete bile duct
ligation. The 3rd group of 39 animals had an in-
complete bile duct ligation. The partial ligation
was relieved in 6 rats and the livers biopsied. The
rats were killed at daily intervals for the first 10
days, weekly for 6 wk. and monthly for 4 months.
The patency of the bile duct was tested at nec-
ropsy and microscopic sections of liver, spleen,
kidneys and adrenals were studied. The hepatic
changes in the rats subjected to complete and
partial ligation of the common bile ducts were
compared. Microscopic changes were observed
as early as 24 hr. in both series. There was a pro-
gressive bile duct proliferation and biliary cirrho-
sis in the rats with complete ligation, with no
animal surviving beyond 2 months. The rats with
partial ligation revealed an early bile duct pro-
liferation, hepatic fibrosis and ‘icteric infarct’
formation. These changes reached their peak at
3 wk. and gradually subsided until only slight
hepatic changes were noted at 4 months. No
appreciable change was noted in the final degree of
hepatic changes in the 3rd group of those animals
relieved of their ligation and those allowed to
continue without release.

1689. Staidies of distribution and localization
of potassium in early myocardial ischemic
injury. Rosert B. JENNINGS,* J. RICHARD
CroutT* anp Wiiuram B. Wartman. Dept. of
Pathology, Northwestern Univ. Med. School,
Chicago, Ill.

The pattern of the loss of potassium from myo-
cardial cells following severe ischemic injury has
been established in dogs using infarct tissue ob-
tained from the posterior papillary muscle of
animals with a high ligation of the circumflex
branch of the left coronary artery. Tissue from this
location was used because it has been found to be
always completely or almost completely infarcted
in animals with a normal vascular distribution,

FEDERATION PROCEEDINGS

Volume 16

and because samples of injured tissue can be
removed for study from this readily identified
site before gross changes of infarction are ap-
parent. Mongrel dogs of both sexes were killed
20 and 40 min., 1, 2, 3, 4, 5, 6, 7, 8, 12 and 24 hr,
following ligation. Tissue samples from the pos-
terior papillary muscle infarct and anterior su-
perior left ventricle (control tissue) were removed
and the potassium content determined by flame
photometry. Tissue prepared by freezing-drying
was stained for potassium by a modification of
MacCallum’s method and the results compared
with the chemical data. The injured fibers lost
potassium slowly for the first 6 hr., and then very
rapidly between 6-8 hr. following ligation, drop-
ping from levels of approximately 75% of normal
to 45% of normal in this time interval. Values of
10-17% of normal were obtained by the end of
24 hr. (This study was supported by grants from
the American Heart Assoc. and Natl. Heart Inst.,
Natl. Insts. of Health, PHS).

1690. Adrenal incorporation of C™ from a
labeled bacterial polysaccharide; influence
of species, route of injection and dosage.
RusseEtt S. JoNES AND YOLANDE CARTER.*
Dept. of Pathology, Univ. of Utah College of
Medicine, Salt Lake City.

After intravenous injection, the C™ from a
labeled polysaccharide of Klebsiella pneumoniae
is concentrated in the adrenal, spleen and liver of
the guinea pig. The adrenal incorporation is the
highest of any tissue on a unit weight basis and
remains in fair concentration at this site for 2
months. Two weeks after a single injection, the
total organ incorporation is 1.2, 0.8 and 10.0% for
the adrenal, spleen and liver, respectively. Re-
spiratory CO, loss occurs only during the first
2 days, while urinary loss occurs throughout the
course of the experiment. By the end of 7 days,
only 20-25% of the total injected polysaccharide
is accounted for on the basis of urinary and CO;
losses. The spleen and liver contain an appreciable
amount of labeled polysaccharide with the same
extraction and monosaccharide characteristics
as the injected material. Autoradiographs reveal
C™ concentration in the cortex of the adrenal
and extraction studies disclose the C to be in the
nonlipid fraction. The rat, mouse and rabbit
have little concentration of C™ in the adrenal
but there is an unusually high concentration of
the label in the lymph nodes of the rat. In com-
parison with the intravenous route, subcutaneous
injections yield only 10-20% as much C* in the
adrenal, spleen and liver of the guinea pig. Varia
tions in administered dose (0.05-10 mg i.v. and
2.0-10 mg s.c.) did not significantly alter the per-
centage incorporation in various tissues of the
guinea pig.

ume 16

san be
ntified
re ap-
killed
| 24 hr,
1€ pos-
ior su-
moved
y flame
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tion of
mpared
rs lost
en very
, drop-
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ulues of
end of
ts from
t Inst.,

rom a
uence
losage.
ARTER,*
llege of

from a
umoniae
liver of
n is the
isis and
e for 2
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).0% for
ly. Re-
she first
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7 days,
-charide
ind CO;
reciable
he same
teristics
s reveal
adrenal
ye in the
rabbit
adrenal
ation of
In com-
taneous
4 in the
». Varit-
i.v. and
the per-
; of the

March 1956

1691. Rékation of sodium, potassium and
chloride intake toe cortisone action. H.
Kaunitz, C. A. SLANETz,* R. E. JOHNSON* AND
J. Guitmain.* Dept. of Pathology and Inst. of
Research in Animal Diseases, Columbia Univ.,
New York City.

Weanling rats were maintained at constant
weight by restricted feeding. A complete purified
diet (30% casein) was used or a similar one vir-
tually free of sodium, potassium and chloride and
supplemented with 0.92% NaCl or equivalent
amounts of CaCls, NH«Cl, NaHCO;, KHCOs,
KCl or no additional salt. When desired, 50 mg of
cortisone acetate was added to each kilogram of
diet. The weekly caloric requirements for weight
maintenance decreased steeply for at least 4 wk.
with diets containing sodium or potassium but
not with those containing chloride in the absence
of sodium or potassium. This decrease was prob-
ably less marked with sodium (especially NaCl).
Values for potassium-containing and control diets
were identical. Addition of cortisone to all sodium-
and potassium-containing diets sharply elevated
the maintenance requirements during the Ist
week; thereafter, the requirements declined
rapidly. Only with NaCl did the requirements
remain elevated. The daily water intakes with
diets containing chloride without sodium or potas-
sium were roughly 40 cc. With either potassium or
sodium with and without cortisone, the intakes
were roughly 10-15 cc, except in the case of NaCl
and cortisone, where the intake was 20 cc. It can
be concluded that the combined action of chloride
and cortisone leads to high caloric requirements
for weight maintenance and high water intake,
effects which are nullified by potassium but are
only mildly counteracted by sodium. The cortisone
action is partly regulated by the dietary salts.

1692. Nutritional properties of molecularly
distilled fractions of autoxidized fats. H.
Kaunitz, C. A. Suanetz,* R. E. Jounson,* H.
B. Knicut,* D. H. SaunpErs* AND DANIEL
Swern.* Dept. of Pathology and Inst. of Research
in Animal Diseases, Columbia Univ., New York
City, and Eastern Regional Research Lab. (Agri-
cultural Research Service, U.S. Dept. of Agri-
culture), Philadelphia, Pa.

The adverse effect on growth and survival of
rats fed autoxidized vegetable oils and lard can
be counteracted by simultaneously feeding fresh
fat (J. Nutrition 55: 577). When drastically au-
toxidized lard and cottonseed oil were molecu-
larly distilled up to 280°C, the residue (nonvola-
tile) fraction, consisting of polymers, exerted a
marked growth-depressant effect (Federation Proc.
14: 408). When the molecularly distilled fractions
were used as the fat source in a rat diet containing
30% casein, growth was nearly as good as with

AMERICAN SOCIETY FOR EXPERIMENTAL PATHOLOGY

521

fresh fat. Thus, the distillate fractions were es-
sentially atoxic. When these fractions were com-
pared with fresh fat as to their protective action
against the growth-depressing effect of polymer,
the protective effect was considerably less than
that of fresh fat. The decrease in protective action
of the unpolymerized fraction of autoxidized
fats could also be recognized without molecular
distillation because the presence of polymer in an
undistilled but autoxidized oil exerted a more
potent growth-depressant effect than did the same
amount of polymer in fresh fat. Thus, molecular
distillation did not cause the effect. The decrease
in protective action was not due to destruction
of polyunsaturated acids by autoxidation as was
shown by compositional analysis of the molecu-
larly distilled fractions.

1693. Intracellular localization of pancreatic
amylase. ANNA Kane Latrp. Div. of Biological
and Med. Research, Argonne Natl. Lab., Lemont,
Til.

In view of the current interest in the role of
the various cell organelles in protein synthesis,
it seemed desirable to investigate the intracellular
distribution of amylase, one of the products of
protein synthesis in the pancreas. Rat pancreas
was studied in the condition of relative rest
produced by overnight starvation and also in the
condition of relatively active synthesis which
follows the secretion of enzyme induced by the
administration of pilocarpine. Homogenates were
prepared in 0.88 mM sucrose and were separated by
differential centrifugation into 5 fractions: the
nuclear, two mitochondrial, a microsomal, and the
final supernatant fluid fraction. After suitable
dilution, these fractions were assayed for amylase
activity by a modification of the method of Meyer
et al. (Helv. Chim. Acta 30: 64, 1947). In each of 6
fractionations of both active and resting glands,
more than 80% of the activity recovered was
found in the supernatant fluid remaining after
sedimentation of the nuclei and mitochondrial
fractions. When the resting glands were frac-
tionated, about 40-50% of the activity remaining
in the supernatant fluid overlying the mito-
chondria was recovered in the microsome fraction
sedimented at 105,000 X g; in the case of the active
glands, under the same conditions, about 60-70%
of the activity was sedimented with the micro-
somes. A much larger proportion of the amylase
activity was sedimented at 105,000 X g when the
supernatant fluid overlying the mitochondria
was diluted with water to bring the sucrose con-
centration to 0.25 m. (Work performed under the
auspices of the Atomic Energy Commission.)

1694. Neoplasms in female Wistar rats occur-
ring spontaneously and following 1000 r

522

anoxic total body irradiation. B. G. Lamson,

L. H. EwEe.u* anp L. R. BENNeETT.* Depis. of

Pathology and Radiology and Atomic Energy

Project, Univ. of California at Los Angeles School

of Medicine, Los Angeles.

Following a single exposure to 1000 r x-rays
delivered under hypoxic conditions of 5% oxygen,
76 female Wistar rats 4 months of age, and 93
nonirradiated rats of the same sex, strain and age
were carefully observed concurrently throughout
their life span. They were autopsied where condi-
tions of tissues permitted. Of 61 autopsied irradi-
ated rats all were dead by 24 months postirradia-
tion, 28 (45.8%) supporting neoplasms. Twenty
nonirradiated control rats were autopsied during
this same 24-month period, with 12 of these (60%)
supporting neoplasms. Fifteen control rats have
subsequently died with autopsy between 24 and
28 months, leaving 28 control rats still alive.
Fourteen of these living rats harbor grossly visible
neoplasms. Including these 14 tumor-bearing
living rats, at least 39 of 63 control rats (61.9%)
have neoplasms. Under these conditions of radia-
tion exposure and in these animals, total body
X-ray irradiation accelerates the appearance of
tumors but does not appear to increase the total
number of tumors when compared with a control
group allowed to live out its natural life span.
(Based on work performed under contract no.
AT-04-GEN-12 between the Atomic Energy Com-
mission and the Univ. of California at Los Angeles
Med. School.)

1695. Deterioration of intermediate com-
plexes between complement and sensitized
sheep erythrocytes. Myron A. LEON (intro-
duced by S. 8S. Hupack). Dept. of Surgical Re-
search, Saint Luke’s Hosp., Cleveland, Ohio.

The deterioration of the complex EAg-p-C’1, 4,2,
the combination of the C’1, C’4 and C’2 of guinea
pig complement (g-p-C’) with sensitized sheep
erythrocytes (EA), has been examined at several
temperatures, using both E.D.T.A. treated
g-p-C’ and pig (porcine) C’3 to estimate the
concentration of the complex. While first order
kinetics were followed in all cases, the activation
energy calculated from the Arrhenius equation
differed, depending on the method of assaying
EAg-p-C’1,4,2. An activation energy of 35,000
cal/m was found, using E.D.T.A. treated g-p-C’,
while an activation energy of 23,000 cal/mM was
found using pig C’3. The deterioration of EAhuC’,
believed to be the complex between human C’1,
C’2 and C’4 and EA was also studied. The rate of
deterioration was about 10 times as rapid as that
of EAg-p-C’1,4,2 and the activation energy of the
reaction was found to be 30,000 cal/m.

1696. Desoxyribose nucleic acid (DNA) -con-
taining cytoplasmic inclusions of human

FEDERATION PROCEEDINGS

Volume 1§

rectal polypoid tumors. CrEcILIE LEUCHTEN-

BERGER. Inst. of Pathology. Western Resery

Univ., Cleveland, Ohio.

Previously, Leuchtenberger (Lab. Investigation,
3: 132, 1954) reported the frequent occurrence of
DNA-containing cytoplasmic inclusions in 63
human rectal polypoid tumors and suggested the
possible viral nature of these inclusions. Examina-
tion of 600 additional cases confirmed the original
observation. Without exception DNA-containing
cytoplasmic inclusions were found in all the
benign and malignant polypoid rectal tumors
examined. They were also present in the liver
metastasis occurring in 2 of the malignant cases
and in polyps of the familial hereditary type,
Comparative studies of different age groups re-
vealed that the number of inclusions found in
polyps of children was strikingly less than in
those of adults. Further cytological and cyto-
chemical investigation demonstrated again the
striking similarity of these inclusions to those
found in known instances of virus diseases, such
as molluscum contagiosum. Phase microscopi¢
examination of Feulgen-stained, osmic acid-fixed
and methacrylate-embedded thin sections of
rectal polyps disclosed a definite structure within
the inclusions. Preliminary electron microscope
studies of rectal polyps done in collaboration with
Dr. Palade at the Rockefeller Institute were
consistent with the interpretation of the viral
nature of these inclusions. (Supported by a grant,
C1814, from the Natl. Insts. of Health, PHS, and
a grant from the Elsa M. Pardee Fndn.)

1697. Comparison of acute pathological ef-
fects of alpha and beta particle radiation of
the rat thyroid gland. Sruarr W. Lippin-
coTtt AND C. J. SHELLABARGER. Med. Dept.,
Brookhaven Natl. Lab., Upton, N. Y.

The selective localization by the thyroid gland
of astatine*"!, element 85, an alpha particle emitter
with a half-life of 7.3 hr., and of iodine!*?, element
53, a beta emitter with a half-life of 2.3 hr., was
utilized to compare the histopathological effects
of these 2 types of radiation following delivery of
known dosages of the isotopes. With 525 ue of I'*
(30,000 rep) occasional stromal edema was noted
in the thyroid gland. With 11 ue of At?! (35,000
rep) stromal edema was always present and with
22 we of At?!! (70,000 rep) there was in addition
evidence of epithelial degeneration in the acini
and sometimes complete individual acinar necro-
sis. Administration of 1050 ue of I'%2 (60,000 rep)
resulted in necrosis of all but a peripheral rim of
acini. Total destruction of all acini occurred
following 44 we At?! (140,000 rep). The degree of
damage to the thyroid gland was not related to
the type of radiation given but appeared to be
correlated quantitatively in terms of equivalent
energy release from each isotope.

t XUM

olume 15

UCHTEN-
Reserve

tigation,
‘rence of
s in 6
sted the
\xamina-
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ntaining
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he liver
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ry type.
oups re-
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e within
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ical ef:
ation of
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. Dept.,

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March 1956

1698. Cortisone-induced alteration in liver
RNA following incubation. CHARLES LOWE
AND RoypEN Ranp.* Children’s Hosp., Buffalo,
BY’.

Polymerized RNA of normal rat liver (NL) is
extractable by hot 10% NaCl solutions (S-NL)
and is subsequently precipitable by ethanol at
20°C for 12 hr. (method I). The precipitate (PP;)
is soluble in water and reprecipitable at 4°C by
5% TCA (PP2). Administration of cortisone (25
mg/day i.m. X 5) produces an alteration of RNA;
the yield from cortisone liver (CL) of PP; is
normal, but that of PP. is 20% of the anticipated.
If S-CL is maintained at 4°C after addition of
ethanol (method II), PP is obtained in normal
yield. Incubation of homogenates of NL at 37°C
results in no loss of PP. with method I or IT;
incubation of CL yields no PP. with method I
and normal amounts with method IT. Incubation
of mixtures of NL and CL results in no PP: with
method I and 120% of expected yield with method
II, PP; and PP» are obtained in normal yield
following incubation of S-NL prior to ethanol
precipitation. Incubation of S-CL resulted in
no yield of PP: with method I, but normal amounts
with method IT. Incubation of equal mixtures
of S-NL and S8-CL resulted in abundant yield of
PP, with either method. These results suggest the
presence of an ‘enzyme system’ in CL, extractable
by hot NaCl solutions, capable of altering physical
properties of RNA. Depending upon experimental
conditions, it acts to increase or decrease yield of
polymerized RNA. An inhibitor of this ‘enzyme
system’ is present in NL-S. (PHS Grants C-1693,
C-2118, American Cancer Society Institutional
Grant.)

1699. Enhancement of proteolysis as mode of
lethal action of ionizing radiation. C. C.
LusHpauGH, L. B. Huaues* anp D. B. HAte.*
Biomedical Research Group, Los Alamos Scientific
Lab., Los Alamos, N. Mex.

A theory to help explain the lethal action of
ionizing radiation is proposed stating that, in
some as yet unknown manner, proteolysis of vital
cells is increased following irradiation by a
disturbance in the normal mechanism antagoniz-
ing and controlling intracellular catheptic activity
and extracellular proteolytic activity. Necrosis
and parenchymatous degeneration appear to be
the morphologic expression of the inability of the
irradiated cell to withstand its uninhibited
cathepsins and to protect itself from the onslaught
of unopposed proteolytic enzymes of the blood
and intestinal tract. The basis for this theory is
found in data revealing early postirradiation
changes: 1) in serum proteolytic inhibitor levels,
2) in acute and chronic susceptibility to paren-
terally injected proteolytic enzyme and 8) in the

E XUM

AMERICAN SOCIETY FOR EXPERIMENTAL PATHOLOGY

523

amount of protection from or enhancement of
radiologic damage by pre- or postirradiation
injections of sublethal amounts of proteolytic
enzymes. Preliminary observations suggest that
parenteral injections of crystalline soy bean
trypsin inhibitor may afford some protection
against radiation damage when injected in the
postirradiation period.

1700. Is the dog a suitable animal for study
of revascularization of the heart? STEPHEN
Mappock. Boston City Hosp., Boston, Mass.
During a study of cardiac revascularization on

the dog, attempts were made to use the spleen as
an added source of blood supply to the heart. To
the writer’s surprise all animals survived ligation
of the descending branch of the left coronary
artery 1 month following the initial operation.
Many of these dogs survived a 2nd ligation of the
right coronary or the circumflex branch of the left
coronary after a period of 4-6 wk. Killing of the
animals and histological studies failed to show
evidence that any benefit could have been derived
from the splenic transplant in many instances
because of a barrier formed by the splenic capsule.
A survey of the literature reveals a striking dif-
ference in results among various groups. The
mortality varies from 10 to 70%. Thirty-six dogs
have been subjected to this procedure. Nembutal
anesthesia plus 100% oxygen connected to a
respirator was used. It would seem that this
technique might foster survival in contrast with
the results of earlier workers who used ether
anesthesia and room air. Various investigators
have pointed out that the dog has numerous
natural intercoronary anastomoses so that one
can reason that, if the initial insult of ligation is
tolerated, a rapid increase of collateral could
occur. Several studies prior to those of the present
writer have raised the question, does the graft
support the heart or does the heart support the
graft?

1701. Reactions in dogs due to hemodialysis
with the Skeggs-Leonards artificial kidney.
F. T. Mauer,* J. V. Youna,* L. C. Watxkins*
AND J. L. Botuman. Mayo Fndn. and Mayo
Clinic, Rochester, Minn.

Many dogs subjected to hemodialysis on the
Skeggs-Leonards ‘artificial kidney’ exhibit an
adverse reaction, consisting of 1) an initial phase
of hypotension, bradycardia and respiratory
depression, 2) an irregularly hypotensive course
and 8) postdialysis depression or coma. Recovery
may follow or death may ensue in 24 hr. or less
after dialysis. At necropsy, subendocardial
hemorrhages are prominent in the left ventricular
wall and degenerative changes have been observed
in the anterior horn cells of the medulla. Minimal
reactions, with no fatalities, were observed in a

524

series of 6 pigs. The toxic reaction has not been
materially modified by varying the procedures
used in preparing the cellophane membranes and
appears to be associated in large part with the
rapid transfusion of the donor blood. Infusions
of norepinephrine have been of both prophylactic
and therapeutic value when given at 0.25-0.5
ug/kg/min. Elimination or marked reduction of
toxic response of dogs to hemodialysis, with
satisfactory to uneventful behavior during and
after the procedure, has been made possible by
1) passing the donor blood through the experi-
mental animal while filling the dialyzer directly
from the arterial cannula, 2) use of isotonic sodium
chloride solution or other noncolloidal solution to
fill the dialyzer or 3) pretreatment of the animal
with adrenergic blockading drugs. For this pur-
pose, piperoxan has been more effective than
phentolamine, azepeptine, Hydergine, Win 8778,
or chlorpromazine. Similar doses of piperoxan
have not modified the anaphylactic response of
sensitized dogs to challenging injections of egg-
white solutions.

1702. Complex of prothrombin conversion fac-
tors. F. D. Mann. Palo Alto Hosp., Palo Alto,
Calif.

Both the stable and the labile prothrombin
conversion factors (cothromboplastic factors)
form complexes with thromboplastin. Another
possible type of intermediate complex in blood
coagulation would be one consisting of the stable
factor plus the labile factor. When the stable
factor (dilute serum) and the labile factor (ad-
sorbed plasma) are mixed and allowed to react
for 10 min. in the presence of calcium and then
lipid thromboplastin is added, high thrombo-
plastic activity is noted almost immediately,
within 30 sec. Coagulation times of 10-12 sec. are
obtained on testing with normal plasma and
calcium, contrasting with times of over 60 sec.
when the preliminary mixing of the stable and
labile factors is omitted and the 2 factors are
simply allowed to react with the lipid thrombo-
plastin fer 30 sec. Preliminary mixing of the lipid
thromboplastin with either factor alone did not
cause appearance of high activity 30 sec. after
adding the missing factor. Only minute traces of
thrombin formed during the preliminary reaction.
Similar mixtures of serum and adsorbed plasma
showed increased labile factor activity when
tested with aged plasma and tissue thrombo-
plastin. Such increased activity disappeared on
removal of stable factor with calcium phosphate,
leaving the labile factor activity initially present.
Recognizing alternative explanations, it is be-
lieved that the simplest basis for the foregoing
observations is a complex of the stable and the
labile prothrombin conversion factors.

FEDERATION PROCEEDINGS

Volume 16

1703. Tissue-localizing gamma globulins in
pathogenesis of human glomerulonephritis,
Rosert C. MELtors anp Louis G. OrTEGa.*
Pathology Div., Sloan-Kettering Inst. for Cancer
Research, and Pathology Labs., Memorial Ctr,
for Cancer and Allied Diseases, New York City.
The principal contribution of this work (see also

Am. J. Path. In press) is to establish beyond a
reasonable doubt that gamma globulins are
localized in the active glomerular lesions in so-
called lipoid nephrosis (chronic membranous
glomerulonephritis) of childhood and in various
types of glomerulonephritis occurring at all ages,
By demonstrating that proteins of the type known
to include antibodies are localized in the active-
lesion sites, the observations fulfill a requirement
essential for the immunoallergic pathogenesis of
these diseases. It is reasonable to think that the
localized gamma globulins are, at least in part,
antibodies against circulating antigens that have
localized in the prospective lesion sites. In con-
ditions where it is thought that the provocative
antigens are of a particular microbial origin, as,
for instance, derived from the hemolytic strepto-
coccus, it may be possible in future work to prove
that the localized gamma globulins are indeed
antibodies against these specific antigens.

1704. Effects of combined exposure to stron-
tium®® and external radiation. W. L. MILNg
AND 8S. H. Coun (introduced by GrorceE C.
Corztas). U. 8. Naval Radiological Defense
Lab., San Francisco, Calif.

The effects of both strontium®® and external
total body x-irradiation administered separately
and in combination were determined in terms of
platelet level changes, body weight and mortality.
Platelet level was found to be the most sensitive
and reproducible index of radiation injury. The
effects produced by radiation from internally
deposited Sr® differed from those resulting from
total body external x-radiation in that both the
onset of radiation damage and the recovery were
more gradual. The depression of platelet level was
proportional to the dose of Sr®*® up to the Lp4/30-
day dose, or approximately 5 yue/gm b. wt. The
depression of platelet level following a sublethal
dose of total body x-irradiation (625 r) was
enhanced in animals previously injected with Sr®
at levels of 0.1 uc/gm or higher. The threshold dose
of Sr injected intraperitoneally required to
produce platelet depression was 0.1 ue/gm b. wt.
The acute depression of platelet level produced
by the simultaneous administration of a sublethal
dose of external radiation and low level of Sr®
was also studied.

1705. Influence of beta-aminopropionitrile
on the development of croton oil pouch.

Qo. THe Ss rere kSlUC

a= + @ @ fe 2. © a o as

—

ans oft za

zs ontoaeowaso a & ge

lume 1§

lins in
»hritis,
RTEGA.*
* Cancer
tal Ctr,
rk City.
see also
yond a
ins are
s in so-
branous
various
ill ages,
> known
active-
irement
nesis of
shat the
in part,
at have
In con-
rocative
igin, as,
strepto-
.O prove
indeed

stron-
. MILNE
RGE C,
Defense

xternal
yarately
erms of
rtality.
ensitive
ry. The
ternally
ng from
oth the
ry were
»vel was
LD 4¢/30-
wt. The
iblethal
r) was
‘ith Sr®
old dose
ired to
1b. wt.
roduced
iblethal
of Sr®

nitrile
pouch.

March 1956

Joun E: Mie.ke,* Joserpu J. Lauicnu anp D.
Murray ANGEVINE. Dept. of Pathology, Univ.
of Wisconsin Med. School, Madison.

Synthetic beta-aminopropionitrile (BAPN) when
fed to rats in the diet may modify the develop-
ment of bone, cartilage and connective tissue, but
the exact mechanism of this modification is not
known. Croton oil pouches, in which fibroblastic
proliferation can be induced within 2-3 wk., were
used as a source of connective tissue. Thirty-three
Sprague-Dawley rats, weighing 161-187 gm, were
divided into groups of 12 control rats and 21
test rats which received between 0.15 and 0.20 ml
of BAPN/100 ml of drinking water/day. After
6, 12 and 18 days, rats from each group were killed
and representative sections from the pouches
taken for microscopic study. It was observed that
test rats gained less weight than controls. The net
weight of the test pouches was less than the
corresponding control pouches; their exterior was
irregular and indistinct from the surrounding
areolar tissue, and, in contrast to the controls, 15
of the test pouches had collapsed by the end of 18
days. In the pouches which collapsed serosan-
guineous fluid was minimal or absent. Micro-
scopically the fibroblasts from the test pouches
showed retarded maturation. The connective
tissue cells were still rounded and vesicular after
18 days, and with Mallory’s trichrom stain there
was demonstrated a persistence of reddish-brown
intranuclear granules at 12 days, whereas they
were diminished in the 12-day control pouches. In
addition, the collagen fibers of the test pouches
did not show the usual progression in develop-
ment or organization with respect to time as did
the collagen fibers from the control pouches.

1706. Mechanism of increased iron absorption
in Tween 20-fed hamsters. H1ipro D. Morr,*
Rosert W. WissLeR, Patricia BARKER* AND
Danute §. Juras.* Dept. of Pathology and
Argonne Cancer Research Hosp., Univ. of Chi-
cago, Chicago, Il.

In previous experiments 5% polyoxyethylene
sorbitan monolaurate (Tween 20) increased the
gastrointestinal absorption of Fe®®, probably from
the cecum and appendix (Proc. Soc. Exper. Biol.
& Med. 86: 170, 1954). In the present study groups
of young adult golden hamsters were fed a syn-
thetic diet, with and without 5% Tween 20, for
8-12 wk. At the end of the diet period, a tracer
dose of Fe®® was given by stomach tube to each
hamster. At intervals of 2-48 hr. the distribution
of Fe5® in segments of the gastrointestinal tract,
with and without content, was measured in the
control and Tween-fed groups. The most sig-
nificant differences in distribution were in the
cecum and appendix. In one experiment, 2 hr.
after the tracer was administered, 80 and 90% of

AMERICAN SOCIETY FOR EXPERIMENTAL PATHOLOGY

525

the total dose of Fe5*® was found in the cecum and
appendix (contents plus wall) of control and
Tween-fed hamsters, respectively. After 24 hr.
the Tween-fed group retained 60% in the cecum
and appendix, while the control group retained
only 20% of the original dose, with most of the
remainder appearing in the feces of the control
group. These results supplemented with serial
barium x-rays of the gastrointestinal tracts of
animals of the 2 groups suggest that administered
iron is trapped and more is absorbed in the cecum
and appendix of Tween-fed hamsters, in spite of
greater gastrointestinal motility and some diar-
rhea in this group. (Supported by Public Health
Service Grants no. C2657 and A374.)

1707. Sterol metabolism in the rabbit. E.
H. Mospacu, E. HALPERN anp J. BRUNDER
(introduced by M. Brvans). Columbia Univ.
Research Div., Goldwater Memorial Hosp., and
Dept. of Medicine, Columbia Univ., New York
City.

Studies were made of the fecal excretion of
sterols and bile acids of rabbits maintained on low
and high fat diets containing known amounts of
cholesterol or 38-cholestanol. On a low fat, sterol-
free diet the animals excreted an average of 50
mg of digitonin-precipitable sterol and 60 mg of
bile acids daily. The addition of 12% triolein to
this diet had no effect on the sterol or bile acid
excretion. The amount of sterol recovered in the
feces of rabbits fed the low fat stock diet, supple-
mented with 1% cholesterol, depended upon the
length of time the animals were maintained upon
the diet..The fecal sterols accounted for 50% of
the intake at the end of 1 wk., and for 90% at the
end of 13 wk. The cholesterol content of both liver
and muscles increased during this period. There
was no increase in bile acid excretion at any time
in response to cholesterol feeding. During the
3rd wk. of balance studies on low and high fat
diets more than 75% of administered 38-cho-
lestanol could be recovered as fecal sterol. At this
time saturated sterols accounted for about half of
the sterol content of liver, muscle and blood.
Tissue storage of 38-cholestanol was increased
when the diet contained added triolein.

1708. Postnephrectomy hypertension of the
dog; potentiation by dietary protein. E. E.
MutrrHeaD. Dept. of Pathology, Univ. of Texas
Southwestern Med. School, Dallas.

One kidney was removed, the mean arterial
pressure was determined for 5-7 days and the
other kidney was removed. The control blood
pressure was 105-125 mm Hg (av. 114). Groups 1,
2 and 8 received 16 ml/kg. daily of physiologic
saline intravenously, an amount of sodium com-
parable to that absorbed through peritoneal

526

dialysis by groups 4 and 5. Hypertension was
considered an elevation of 25 mm Hg or more
(range +25-80, av. +41 mm Hg). The average
weight was 10.6 kg. The experiment lasted 4 days;
tissues were studied microscopically. Group 1
(21 dogs), received no diet: 4 of 21 dogs developed
hypertension. Group 2 (10 dogs), diet with no
protein and no electrolytes (40 ml/day, 130 cal.,
52% fat, 48% carbohydrate, vitamins): 1 of 10
dogs became hypertensive. In groups 1 and 2, 5 of
31 dogs became hypertensive. Group 3 (25 dogs),
diet containing protein (40 ml, 250 cal., 27% fat,
26% CHO, 47% protein, vitamins): 17 of 25 dogs
became hypertensive. Group 4 (19 dogs), same diet
as group 3 plus daily peritoneal dialysis; 17 of 19
dogs became hypertensive. Group 6 (11 dogs),
protein diet with additional calories (300 ml/day,
550 and 700 calories, fat 17 and 45%, CHO 59 and
35%, protein 24 and 20%), plus peritoneal dialysis;
all 11 dogs became hypertensive. In groups 3, 4
and 6, 45 of 55 dogs became hypertensive. Cardio-
vascular lesions were more prominent in groups 3,
4 and §. Dietary protein potentiates the hyper-
tension of bilateral nephrectomy.

1709. Arteriosclerosis in pyridoxine-deficient
monkeys and dogs. CHARLES W. MusHETT
AND Guapys A. Emerson. Merck Inst. for Thera-
peutic Research, Rahway, N. J.

The observation of Rinehart and Greenberg
(Am. J. Path. 25: 481, 1949) that arteriosclerotic
lesions develop in pyridoxine-deficient monkeys
has been confirmed and extended in Rhesus
monkeys and dogs. Diets employed included that
of the authors cited above as well as our own
synthetic rations. Fifteen monkeys subjected to
pyridoxine deficiency for periods of 4-14 months
exhibited arteriosclerosis grossly or micro-
scopically in the lower abdominal aorta and iliac
arteries. In addition to lesions in the larger
vessels, these monkeys have shown arteriosclerosis
microscopically in one or more of the following
organs: heart, kidney, pancreas, testis, ovary,
uterus, thymus, liver, adrenal, colon and lung.
The artgriosclerosis is primarily an intimal fibrosis,
variably associated with an increase in meta-
chromatic ground substance and degeneration
and duplication of the internal elastic lamella.
Medial changes are sometimes present. Sudanophi-
lic material has not been observed in these lesions.
In a small series, addition of 2% cholesterol to the
pyridoxine-deficient diet did not cause deposition
of Lipoidol or anisotropic material in the arterial
lesions of monkeys. Repeated subcutaneous
injection with the pyridoxine antagonist, desoxy-
pyridoxine, effected arteriosclerosis in the monkey
maintained on a mixed stock ration. In pyridoxine-
deprived dogs, arteriosclerosis has been observed
grossly in the lower abdominal aorta and/or the

FEDERATION PROCEEDINGS

Volume |

ascending aorta. Other pathologic findings j
deficient animals include: relative enlargement @
liver, kidneys, heart, adrenals, thyroid an
pituitary, atrophy of thymus and lymph node,
fatty metamorphosis in liver and kidneys, hyper
trophy and hyperplasia of pancreatic islets,
thinning of adrenal glomerulosa and _ dental
abnormalities.

1710. Rapid test for thymolytic activity of
corticoids in nestling rats. E. M. NADEL aw
A. G. Hinear.* Natl. Cancer Inst., Natl. Insts,
of Health, Bethesda, Md.

The sluggishness of the pituitary-adrenal system
in the newborn rat has been utilized for a 3-day
bioassay of ACTH, based on thymus weight
reduction (Bruce, PARKES AND Perry. Lance
262: 790, 1952). Significant thymus weight re-
duction occurs within 16 hr. after one injection of
cortisone in adrenalectomized mice (PASCHKIS.
Rec. Prog. Horm. Res. 8: 113, 1953). Utilizing both
reports, we have developed an overnight thymo-
lytic test for corticoids in 10-14-day-old intact
rats. Ten litters of the same age, each containing
at least 7 animals (19-25 gm), are divided so that
1 animal from each litter is used in each of 7 assay
groups (10 animals/group) to include an unknown
at 3 dose levels (or 3 unknowns), 1 vehicle control
and cortisol at 3 dose levels (100, 200, and 400).
The total of the body weights of the 10 animals
in each of the 7 assay groups is kept within a
range of 5 gm. Dried standards and unknowns are
dissolved in warmed absolute ethanol and diluted
to 10%. (Alternatively they may be dissolved in
acetone, made up to volume with corn oil, sesame
oil or propylene glycol, following which the
acetone is allowed to evaporate.) One-tenth
millimeter is injected subcutaneously at 4-5 pM.
All animals are chloroformed 17 hr. later and
weighed; thymus glands are dissected immediately
(under magnification) or after refrigeration (up to
4 hrs., 4°C), and thymus:body weight ratios
tabulated and plotted. Two workers (working 3 hr.
each) can conveniently compare in a 24-hr. period
the thymolytic activity of an unknown with that
of cortisol and controls without the necessity of
adrenalectomy, postsurgical care or special diets.
The semilog-dose:response of cortisol is linear.
By this test metacorten had 3-4 times the activity
of cortisol.

1711. Comparison of ability of scorbutic and
control guinea pigs to metabolize large
doses of administered cortisol. E. M. Nap&l
AND S. Burstern.* Natl. Insts. of Health, Be-
thesda, Md., and the Worcester Fndn. for Expl.
Biology, Shrewsbury, Mass.

Despite the presence of increased amounts of
corticoids in the blood and urine of scorbuti¢

gu
liv

X-]

ae
| -<

tre

E XUM

Volume |

idings j
zement of
roid ana
oh nodes,
rs, hyper.
ic islets,
d dental

tivity off
ADEL AND
utl. Insts,

al system
r a 3-day
s weight
r. Lanecel
eight re-
ection of
»ASCHKIS,
zing both
t thymo-
ld intact
mntaining
d so that
of 7 assay
unknown
e control
ind 400).
) animals
within a
Owns are
d diluted
olved in
|, sesame
1ich the
ne-tenth
4-5 PM.
iter and
.ediately
ym (up to
t ratios
cing 3 hr.
r. period
rith that
essity of
al diets.
; linear.
activity

itie and
e large
. NADEL
uth, Be-
yr Expl.

ounts of
corbutie

March 1956

guinea pigs, such animals are unable to deposit
liver glycogen as efficiently as do controls. More
recent studies utilizing radioacetate have indi-
cated that there is an increased uptake of labeled
acetate not only into adrenal cholesterol but also
into adrenal cortisol and corticosterone. This has
suggested that there may be both overproduction
and underutilization of corticosteroids in scurvy.
Further study of the over-all metabolic efficiency
of the scorbutie guinea pig to utilize cortisol was
undertaken by administering a large dose of corti-
sol (100 mg p.o.; 25 mg i.p.) to scorbutic and
control guinea pigs, and then quantitatively
measuring and isolating the cortisol excreted in
the urine within the ensuing 24 hr. The scorbutic
guinea pigs excreted significantly greater amounts
of cortisol than did control animals. These findings
suggest that cortisol is metabolized relatively less
efficiently in scurvy.

1712. Survival of bacteria in phagocytes from
normal and x-irradiated mice. Eric L. NEL-
son,* JoAN R. BecKER* AND C. PutLurp MILLER.
Dept. of Medicine, Univ. of Chicago, Chicago, Ill.
The ability of phagocytosed bacteria to survive

within phagocytic cells was examined in vitro
employing peritoneal exudates from normal and
immunized mice exposed to various doses of
x-radiation. Exudates were induced by injection,
intraperitoneally, of 2 ml 15% gelatin, followed
16 hr. later by: intraperitoneal injection of a cold
suspension of washed bacteria, 108 Pseudomonas
aeruginosa. Exudates were harvested, washed
twice in heparinized Simm’s solution, and sus-
pended in Fischer’s V-614 medium to a concen-
tration of 106 cells/ml. The Fischer’s medium
contained sufficient Polymyxin B to kill any
extracellular bacteria. Samples withdrawn peri-
odically were incubated with succinate and
triphenyltetrazolium chloride (TTC), smeared,
fixed in methanol, stained with light green and
examined microscopically to determine: the types
of cells, the percentage containing living bacteria
and the average number/cell. Reduction of TTC
by intracellular bacteria was the criterion of
viability, which was confirmed by cultural meth-
ods. The intracellular bacteria survived the
presence of the antibiotic in the suspending
medium. Results indicate that bacterial multipli-
cation occurred within phagocytes obtained from
irradiated mice but not in those from normal
mice, nor irradiated mice previously immunized
by multiple subcutaneous and intraperitoneal
injections of heat-killed Ps. aeruginosa.

1713. Distribution and localization of tissue
antigens as determined with fluorescent
antibody to rat lymphosarcoma. WILBUR
Fiske Noyres* anp Maurice M. Rapport (in-

AMERICAN SOCIETY FOR EXPERIMENTAL PATHOLOGY

527

troduced by C. P. Ruoaps). Div. of Expil.

Pathology, Sloan-Kettering Inst. for Cancer Re-

search, Sloan-Kettering Div., Cornell Univ. Med.

College, and the Div. of Labs. and Research, New

York State Dept. of Health, New York City.

Antiserum prepared against the mitochondrial
fraction of rat lymphosarcoma (Rapport ef al.
Cancer 8: 538, 546, 1955) was labeled with fluo-
rescein (Coons. J. Exper. Med. 91: 1, 1950),
absorbed with lyophilized liver and kidney, and
used to stain frozen sections of normal and neo-
plastic rat tissues. Lymphosarcoma, intestine,
thymus and skin stained strongly, while spleen
and brain gave weaker reactions. Liver, kidney,
heart, adrenal and lung gave no reaction. Thus,
the staining cannot be due to the connective tissue
antigen of Cruickshank and Hill (J. Path. & Bact.
66: 283, 1953). Sarcoma stained uniformly; the
intestinal staining was limited to the epithelium
of the villi, and the staining of the brain to oc-
sasional neurons. Since no staining was observed
with liver, kidney and lung, the results indicate
that epithelial cells of different organs may be
differentiated by this method. In all cases staining
was limited to the cytoplasm of the cell.

1714. Chromosomes of Yoshida ascites tumor
cells. Susumu OHNO* AND Riosgun KinositTa.
Dept. of Cytology, Med. Research Inst., City of
Hope Med. Ctr., Duarte, Calif.

The authors have reported previously on karyo-
types of rat diploid lymphoblasts, especially
referring to 3 chromosomes with secondary con-
strictions or nucleolar organizers. Following
these studies, Yoshida ascites sarcoma cells have
been studied similarly. Although widely variable,
the chromosome number frequently found was
near 40. Ten subdiploid, late metaphase figures
were selected to investigate structural details of
the chromosomes. Their karyotypes were all
similar but differed from those of the lympho-
blasts. A large, V-shaped, mid-centric chromo-
some, described by Makino, was always present,
whereas the largest pair of metacentric chromo-
somes observed in the lymphoblast was not found.
In addition to 3 metacentric chromosomes with
secondary constrictions, morphologically corre-
sponding to those of the lymphoblast, a mid-
centric chromosome at the 4th or 5th place in the
chromosome alignment was found to have a
secondary constriction at one of its subterminal
regions. Increase in number of chromosomes with
secondary constrictions appears to coincide with
an increase in volume or number of the nucleoli in
the tumor cells. It is still questionable, however,
that the appearance of the additional chromo-
somes with secondary constrictions is associated
with malignancy. The chromosomal changes
observed in the tumor cells, namely the ap-

528

pearance of certain chromosomes of nonhost
origin and the absence of certain chromosomes of
host origin, at least suggest that these cells are a
type which differ from normal host somatic cells.

1715. Antibody localization in nephrotoxic
nephritis. Louis G. ORTEGA* AND RoBeErt C.
ME tutors. Cytochemistry Section, Sloan-Ketter-
ing Inst. for Cancer Research, New York City.
Thirty rats were killed at intervals up to 3

months after receiving multiple intravenous in-

jections of nephrotoxic globulins, with moderate
to severe nephritis developing in all as judged by
proteinuria and histologic changes. No abnor-
malities were detected in 8 untreated controls or
in 3 rats receiving normal rabbit serum. Frozen
sections of rat kidneys, representing every interval
at which an animal was killed, were stained with
antibodies that were prepared against rabbit
y-globulin in the goat and rat y-globulin in the
rabbit, and coupled to fluorescein by the method

of Coons and Kaplan (J. Exper. Med. 91: 1, 1950).

Fluorescence patterns were studied visually and

photometrically, and their specificity evaluated

with each stain by blocking the reactions with un-
coupled antibody and by staining with fluorescein-
coupled rabbit antihuman y-globulin. Quantita-
tive data on relative concentrations of fluor in the
stained section was obtained by comparing the
fluorescence intensity of the glomeruli with that
of the tubules. Nephrotoxic antibodies were found
to localize selectively in the basement membranes
of the glomerular tufts throughout the experi-
mental period. Autogenous (rat) antibodies were
also demonstrated in the tufts exclusively, but in
2 statistically distinct orders of concentration,
viz low levels, as estimated by fluorescence
intensity, in the first 6 days after injection—
although still significantly above that in the
control kidneys—and high levels thereafter. The
remarkable persistence of antigenicity of the
injected, localized foreign antibody suggests the
feasibility of similarly demonstrating localized
antigens in long-standing, active human nephritis.

1716. Serum protein changes in postnecrotic
cirrhosis produced by ethionine. Hans
Popper, ALVIN DuBIN,* DANIEL S. KusHNER,*
GeEoFFREY KENT,* CLARA BrRucE* AND ELAINE
Herzoa.* Hektoen Inst. for Med. Research,
Cook County Hosp., Chicago, Ill.
Administration of ethionine to rats for more

than 4 months causes a coarse nodular cirrhosis

characterized by large regenerative nodules, foci
of cholangiolofibrosis, focal necrosis, bizarre re-
generation of the liver cells and splenomegaly.

The alterations are more uniform with continuous

0.3% ethionine diet than if an 0.5% ethionine diet

is alternated with stock diet. Rats with post-

FEDERATION PROCEEDINGS

Volume 16

necrotic cirrhosis, which developed from multiple
continuous focal necroses, showed increased serum
gamma globulin, measured by electrophoresis and
turbidimetry, to an average of almost twice that
of the norm; simultaneously, the serum albumin
level decreased to almost half the norm. The
fraction corresponding to beta globulin also
revealed a significant rise, alpha globulin ap-
parently remaining unaltered and the serum total
protein only slightly reduced. Such serum protein
changes are only slight in rats after 6 wk. on
ethionine diet, in which, nevertheless, diffuse
hepatocellular injury with interstitial infiltration
is associated with decreased hepatic total protein,
albumin, esterase and elevated phosphatase, all
to a degree similar to the long-term experiments.
The serum protein changes thus did not parallel
the morphologic and biochemical degree of liver
cell damage but rather the degree of cirrhosis
formation. They are not prevented by splenec-
tomy and are not associated with bone marrow
plasmacytosis. The gamma globulin elevation in
the experimental postnecrotic cirrhosis which
simulates that of the human lesion is thus con-
sidered the direct result of hepatic alterations.
(Supported by Grants no. C-2030 and A-334,
Natl. Insts. of Health, PHS.)

1717. Hepatitis in hamsters inoculated with
equine abortion virus: development of in-
clusions and growth cycle. CHARLES C.
RANDALL AND E. C. Bracken.* Dept. of Micro-
biology, Vanderbilt Univ. School of Medicine,
Nashville, Tenn.

Equine abortion virus produces a striking
hepatitis in hamsters, culminating in death in
18-24 hr. A characteristic intranuclear inclusion
appears in practically all parenchymal cells. The
constancy of the infection and nature of the
lesion indicate that the hamster liver is an ex-
cellent medium for the study of a specific animal
virus. Three-week-old hamsters were inoculated
intraperitoneally with 1.0 ml of a suspension of
hamster liver containing approximately 10° LD.
Livers were collected over a period of 15 hr. at 3-hr.
intervals for histological, histochemical and cell
fractionation studies. Blood also was collected for
viral titrations. The present report is restricted to
observations of the growth cycle and develop-
ment of inclusions. Sequential morphological
changes will be illustrated. Uniquely, typical
inclusions (type A) were noted in a small number
of cells at 6 hr., whereas at 9-12 hr., and there-
after, more than 99% of the parenchymal cell
nuclei contained inclusions. The great majority
were filled with homogeneous inclusion material
in contrast to the small number which contained
shrunken type A bodies. Corresponding Feulgen
stains showed the various sequential forms to be

PS ae Sat Fee ee ed

Din ne Baek

ume 15

ultiple
| serum
sis and
ce that
lbumin
a. The
n also
in ap-
n total
protein
wk. on
diffuse
tration
rotein,
ase, all
iments.
parallel
of liver
irrhosis
plenec-
marrow
tion in

which
us con-
rations.
A-334,

d with
of in-
Les C.
’ Micro-
edicine,

striking
eath in
rclusion
lls. The
of the
- an ex-
, animal
culated
nsion of
10° LD5o.
_at 3-hr.
and cell
cted for
ricted to
develop-
ological
typical
number
d there-
mal cell
majority
material
ontained
Feulgen
ms to be

March 1956

positive and thus presumably contained DNA.
For comparison, LDs5o titers of blood and liver are
shown. Both reach a maximum between 9 and 12
hr. and correlate with the development of in-
clusions. (Aided by a grant from the Grayson
Fndn.)

1718. Gastric secretion of basic dyes. F. E.
Ray (introduced by MicuaEt Kuen). Cancer
Research Lab., Univ. of Florida, Gainesville.
Ray and Jung (Brit. J. Cancer 5: 358, 1951)

proposed a formula to correlate gastric secretion

of basic dyes with their ionization constants. The
data for secretion were obtained from the paper

by Ingraham and Visscher (J. Gen. Physiol. 18:

695, 1935). Since both the values for ionization

constants and gastric secretion left much to be

desired, Woislawski (Proc. Soc. Exper. Biol. &

Med. 82: 152, 1953), of this laboratory, carried out

an extensive investigation into the former, and

the present paper reports results on the latter.

The previous data on gastric secretion reported

the ratio of dye concentration in gastric juice to

that in blood. In this laboratory, Klein, Cabrera
and Argus (Am. J. Physiol. 180: 655, 1955) showed
that the concentration of dye in the blood changes
very rapidly; this would create an uncertainty
in the ratio. The present study was carried out
with guinea pigs bearing gastric fistulas described

by Breidenbach and Ray (Am. J. Physiol. 180:

637, 1955). The animals were stimulated with

histamine and the dye collected for 2 hr. or until

no longer detectable. The total secretion was
compared with the administered dose. As pre-

dicted, there is an optimum pxka of between 9.0

and 10.0; higher or lower values give decreased

secretion. Methylene green, neutral violet, tolui-
dine blue and thionin show a decreasing order of
secretion. Previous work gave increasing ratios,
which our data suggest were dependent on dif-
fusion rates. (Supported by a grant from the
Damon Runyon Fund.)

1719. Rapid production of plasmacytoses in
rabbit lymph nodes. J. W. Resuck, A. H.
Vevtpuuis* AND L. A. SwWINEHART.* Depts. of
Labs. and Obstetrics and Gynecology, Henry Ford
Hosp., Detroit, Mich.

Ten cubic centimeters of nonradioactive gold
suspension (Abbott) of 0.003-u particle size con-
taining hyaluronidase (100 vu) were injected into
the left lower thoracic mammary gland of 6
rabbits. At 6, 12, 24 and 48 hr. and at 1 wk. imprint
and section studies were made of the ipsilateral
internal mammary and axillary lymph nodes. One
thousand-cell differential counts of lymph nodes
of 4 control rabbits revealed plasma cells present
(range 0.65-1.25%). Gold-injected animals re-
vealed marked increases in plasma cells in their

AMERICAN SOCIETY FOR EXPERIMENTAL PATHOLOGY

529

regional lymph nodes: 4.8% at 6 hr., 6.3% at 12
hr. and 22.2% at 24 hr. Lymphocytes and reticu-
lum cells served as dual sources of such plasma
cells. Some plasma cells presented unusual nuclear
budding and cytoplasmic vacuolation. An ad-
ditional 4 rabbits were similarly injected with
radioactive gold suspension. After determination
of rate of transport of radioactive gold to regional
lymph nodes from the breasts and the concentra-
tion of radioactive gold at timed intervals, the
lymph node reactions were studied at 1, 2, 4 and
12 wk. The customary late plasma cellular in-
creases due to radiation were confirmed.

1720. Substances released from the skin fol-
lowing thermal injury: Histamine and
Proteins. Sot Roy RosenTHAL, CHARLES
Samet,* Ricwarp J. WINZLER AND SELWYN
SuKounik.* Instn. for Tuberculosis Research,
Univ. of Illinois and Depts. of Preventive Medi-
cine and Biochemistry, Univ. of Illinois College
of Medicine, Chicago.

A method is described for obtaining diffusate
directly from burned skin of the dog circumvent-
ing the circulation. Split thicknesses of skin are
secured about glass cylinders so that the cut
surface is in continuity with the inside of the
cylinder. The outer aspect of the skin is burned
by immersing in hot water and the materials
diffusing from the skin are collected into a saline
solution within the tube. It was found that
exposure to heat caused the liberation of histamine
into the diffusates of the skin. The amount of
histamine liberated was dependent upon the time
and the temperature of the burning. Likewise, the
release of protein was increased with the time and
temperature of burning. Determination of the
hydroxyproline content and the electrophoretic
behavior of such preparations indicated that the
major portion of the proteins released resembled
gelatin. In addition, a smaller amount of other
proteins containing appreciable amounts of hexose
and ‘sialic acid’ was released, indicating that
glycoproteins were likewise liberated. (Aided in
part by a contract (NR 114 161) between the Office
of Naval Research, Dept. of the Navy and the
Univ. of Illinois.)

1721. Effect of Hufnagel valve on susceptibil-
ity of dogs to endocarditis. JosEPH RosuHE,
BENJAMIN HIGHMAN AND PauL D. ALTLAND
(introduced by R. D. Liuu1e). Clinic of Surgery,
Natl. Heart Inst. and Natl. Inst. of Arthritis
and Metabolic Diseases, Natl. Insts. of Health,
Bethesda, Md.

A method has been reported for producing acute
and subacute bacterial endocarditis by intra-
venous injections of Staphylococcus aureus and
Streptococcus mitis, respectively, in dogs with

530 FEDERATION
aortic insufficiency. All dogs with aortic insuf-
ficiency which received either a full course or a
single 1 ce intravenous injection of culture de-
veloped endocarditis. None of a series of normal
controis showed endocarditis. Hufnagel valves
were placed in 5 dogs with aortic insufficiency to
study the effect of a reduced cardiac workload on
susceptibility to endocarditis. One dog received 4
daily injections of Staph. aureus, while another
received a single 1 ce injection. Both dogs de-
teriorated rapidly and died within 6 days. Autop-
sies revealed acute necrotizing endocarditis with
subendocardial hemorrhages; hemorrhagic and
suppurative lesions and infarcts were noted in the
viscera. One dog received 9 daily injections of S.
mitis, while the remaining 2 received a single 1 cc
injection. They were killed at 10-14 days; at
autopsy, 2 showed large aortic and mitral vegeta-
tions and visceral infarcts. One dog which received
1 ce of S. mitis showed no evidence of active endo-
carditis. The dogs with endocarditis often de-
veloped bacterial vegetations at the suture line
and tube-vessel junctions in the aorta. The
Hufnagel valve afforded no striking protection
against endocarditis in these dogs. This is at-
tributed largely to persistence of coronary insuf-
ficiency, valvular deformity and residual aortic
insufficiency.

1722. Bioassay of human hypophysis for thy-
rotropic hormone correlated with cytology
and hormone therapy. AGNES B. RusSFIELD
AND SHOLEM PosTEL (introduced by BENJAMIN
CastTLEMAN). Pathology and Medicine Depts.,
Massachusetts General Hosp., Boston.

The hypophysis was removed at autopsy from
16 patients (7 female and 9 male) ranging in age
from 4 to 88 yr. who died of a variety of diseases.
The anterior lobe was divided sagittally into two
equal parts, one of which was studied histo-
logically. The other was frozen in dry ice, then
assayed for thyroid-stimulating activity. The
hypophyses of the majority of the patients who
had received no hormone treatment were of ap-
proximately equal potency in their ability to
stimulate I'*! uptake by the thyroid of the day-
old chick. In 2 patients treated with cortisone for
lupus erythematosus and carcinoma of the breast,
respectively, the amount of thyroid-stimulating
activity per unit weight of anterior lobe was
reduced. An unusually high level of thyroid-
stimulating activity was found in 3 patients, all
of whom had anatomical evidence of decreased
gonadal activity, but not in 1 myxedematous
patient who had received terminal thyroid medica-
tion. The concentrations of apparent thyroid-
stimulating activity could not be correlated with
the age or sex of patients in this small series. These
limited observations are consistent with the idea
that in man, as in animals, treatment with corti-

Volume 18

PROCEEDINGS

sone may decrease the amount of thyrotropic
hormone produced by the hypophysis.

1723. Effect of total body x-irradiation on the
parakeet. Hans G. ScHLUMBERGER AND UL-
rRicH K. HENScHKE.* Depts. of Pathology and
Radiology, Ohio State Univ., Columbus, Ohio.
The effect- the parakeet, Melopsittacus

undulatus, of a single dose of total body x-irradia-

tion was studied in 325 birds. With x-rays of 250

kv and a half-value layer of 1.8 mm copper the

LDso in 30 days was found to be 1800+75 r. Nine

dose levels ranging from 500 to 3000 r were em-

ployed; at each level the great majority of deaths
occurred 10-15 days after irradiation. These birds
were not molested in any way, but in an additional
group of 25 birds subjected to 2000 r the peripheral

blood was examined. The white blood cell count 4

days after exposure was only } of normal; 10-14

days after irradiation these cells had almost

wholly disappeared from the circulating blood.

Depression of the erythrocyte count was also

most marked 2 wk. after exposure when it had

dropped to 3 of normal. The marrow was aplastic,
with only occasional nests of reticulum cells re-
maining. Recovery began during the 3rd wk. after
irradiation. At autopsy hemorrhages were rarely
encountered, the outstanding lesion in addition
to the aplastic marrow was usually an acellular

pneumonia frequently associated with the pres-
ence of Aspergillus. The cause for the greater
resistance to irradiation of this bird as compared
with mammals has not been determined, but the
fact that in birds the circulating erythrocytes and
thrombocytes are intact nucleated cells rather
than the effete erythrocytes and platelets found in
mammals may be significant.

1724. Interaction of nucleic acid with some
cationic dyes. Mertvin D. ScHOENBERG,*
CHARLES LOESER* AND JAMES L. ORBISON.
Dept. of Pathology, Univ. of Rochester School of
Medicine, Rochester, N. Y., and Dept. of Anat-
omy, Western Reserve Univ. School of Medicine,
Cleveland, Ohio
The interaction of nucleic acid (DNA) with

several diamino-acridine and thiazine dyes has

been studied in terms of the metachromatic and
fluorescent properties of the dye-DNA complex
in vitro and in vivo. Unlike many of the anioni¢
polyelectrolytes which function only as positive
metachromatic substrates, DNA can induce both
positive and negative metachromatic transitions.
Consideration of the effects of temperature and
ionic strength on the 2 metachromatic regions
indicates that 2 distinct types of DNA-dye com-
plexes are formed. The stability of the negative
metachromatic complex is much greater than the
positive complex. While the positive complex
results in fluorescent quenching, the formation of

irr
of.
be

the
ter
size
stu
tiss
cho
esti
blo
the
wou
and
at 1
Ato

me 16

tropic

n the
p UL-
y and
hio.

ittacus
radia-
of 250
er the
. Nine
re em-
deaths
> birds
itional
pheral
ount 4
- 10-14
almost
blood.
s also
it had
lastie,
alls re-
:. after
rarely
idition
ellular
> pres-
yreater
npared
nut the
tes and
rather
yund in

1 some
BERG,*
RBISON.
chool of
f Anat-
edicine,

) with
ves has
tic and
omplex
anioni¢
ositive
ce both
sitions.
ire and
regions
ye com-
egative
han the
»omplex
ation of

March 1956

the negative complex results in a markedly in-
creased fluorescent yield. The differences in the
metachromatic and fluorescent behavior is not
entirely a function of the molecular weight of the
DNA. It appears that the differences are more
intimately related to modifications of the physico-
chemical state of the DNA. The in vitro behavior
can be demonstrated in vivo.

1725. Intramolecular organization of acetyl-
cholinesterase as shown by irradiation
studies. IRvING SERLIN* AND GEORGE C.
Corzias. Med. Dept., Brookhaven Natl. Lab.,
Upton, N. Y.

Acetylcholinesterase preparations were exposed
to cobalt-60 gamma rays in order to determine
whether effects on submolecular units of the
enzyme were demonstrable by analyzing the
residual enzyme activities. Dilute preparations of
partially purified electric eel organ acetylcholin-
esterase were irradiated with 630,000 r. Several
properties, e.g. energy of activation, substrate-
induced inhibition, ete., of the residual activities
and of the nonirradiated control were found com-
parable except for a marked heat sensitivity of the
irradiated samples. Apparently those molecules
surviving the irradiation were damaged suf-
ficiently to be more susceptible to heat. Likewise,
the inactivation of dry samples by irradiation
indicated that eel acetylcholinesterase activity
was not dependent on the integrity of the entire
molecule. These preparations, either from par-
tially purified extracts or whole tissue, had
identical inactivation rates which permitted
calculation (after E. Potuarp) of the fraction of
molecular mass destroyed by single ionizing
radiation events. This radiosensitive mass is of
interest since it approximates a unit capable of
independent activity. Basing our calculations on
irradiation data obtained at 18°C., the target mass
of eel organ acetylcholinesterase was estimated to
be 235,000. Since the molecular weight is 3,000,000,
the radiosensitive submolecular unit (as de-
termined by this method) is approximately ;'s the
size of the macromolecule. Initial irradiation
studies were also carried out on 3'mammalian
tissues. The radiosensitive unit mass of acetyl-
cholinesterase in each of these was tentatively
estimated to be: rat brain, 74,000; human red
blood cells, 306,000; human plasma, 210,000. Thus,
the radiosensitive acetylcholinesterase unit mass
would appear characteristic of the tissue source
and cannot be correlated with enzyme specificity
at this time. (This work was supported by the
Atomic Energy Commission.)

1726. Agents influencing isoproterenol-in-
duced hypotension. JEAN Sicé aND JULES
M. Sevetz (introduced by I. Davipsoun).

AMERICAN SOCIETY FOR EXPERIMENTAL PATHOLOGY

531

Dept. of Physiology and Pharmacology, Chicago

Med. School, Chicago, Ill.

Atropine and Dibenzyline or piperoxan do not
alter the hypotensive response to isoproterenol,
although atropine induces a secondary hyper-
tensive phase. Hexamethonium considerably
augments the hypotension induced by the drug.
Increasing doses at procaine (1-20 mg pk) corre-
spondingly increase the response (20-45 mm Hg)
to a standard dose of isoproterenol. Hydergine
decreases the usual response to the drug; a normal
reaction can, however, be restored by administra-
tion of ergotamine. An inhibition of the ergot
alkaloids by their dihydro derivatives has been
reported (RoTHLIN AND CrERLETTI, 1950). The
isoproterenol-induced hypertension in ergotized
animals (LANDS, TAINTER et al. 1950) is reversed
by procaine and prevented by urethane anesthesia.
Urethane, however, does not affect the decrease
induced by methacholine, neostigmine (FRom-
HERZ, 1946) or some antihistaminic agents of the
isoproterenol hypotension. This work was done in
mongrel dogs of either sex, all drugs being in-
jected intravenously.

1727. Effects of anterior hypophysis on osteo-
arthritis in underfed mice. MARTIN SILBER-
BERG AND RutH SILBERBERG. Snodgras Lab.,
City Hosp. Div., and Dept. of Pathology, Wash-
ington Univ., St. Louis, Mo.

Anterior hypophyseal transplants and extracts
increased the incidence and advanced the onset of
osteoarthritis in susceptible rodents (Am. J.
Path. 15: 547, 1939; Endocrinology 54: 26, 1954),
while underfeeding inhibited and retarded the
evolution of osteoarthritis (Growth 13: 359, 1949).
In order to determine the role of food intake in the
effects of hypophyseal hormone on the joints,
pituitaries were transplanted into underfed mice;
108 strain A males were fed Purina Laboratory
Chow restricted to 60% of the ad libitum con-
sumption. This diet maintained a body weight of
22 gm as compared to a normal weight of about 30
gm. Fifty-two 1-month-old mice received trans-
plants of 4 anterior pituitaries obtained from
closely related young donors; the remaining 56
mice did not receive transplants. The incidences
of osteoarthritis observed are given below together
with those seen in ad libitum fed controls and in
ad libitum fed mice bearing hypophyseal grafts.

Mean Age of Osteoarthritis

All Mice Mean age
Experiment (mo.) % (mo.)
Untreated 19.5 41 18.3
Underfed 15.2 25 22.5
Untreated + pit. 17.6 80 16.0
Underfed + pit. 14.7 54 14.1

The injurious effects of anterior hypophyseal
secretions on the joints are thus present also in
underfed animals and are therefore within limits

532

at least independent of the amount of food con-
sumed. (Aided by Public Health Service Grant
A-22 C5.)

1728. Ability of spleen to protect against
leukemia in mice. Eric L. Srmmons (intro-
duced by L. O. JacoBson). Argonne Cancer
Research Hosp., Univ. of Chicago, Chicago, Ill.
CF no. 1 mice, which are not susceptible to the

lymphatic leukemia found in the DBA/2 mouse,

will develop leukemia if they are irradiated
previous to inoculation, the degree of mortality
depending upon the magnitude of the exposure.

When such nonsusceptible mice are spleen-

shielded during exposures ranging between 100

and 600 r, they do not develop leukemia following

inoculation. Preliminary results indicate that
removal of the spleen after as short an interval as

15 min. still results in substantial ability of the

body to protect itself against the invading leu-

kemic cells. Injection of cell preparations of
spleen, liver and bone marrow to the totally
irradiated body, followed by inoculation with
leukemic cells at varying intervals, shows that
such injections immediately after x-irradiation
are not as effective as the presence of a shielded
portion of the animal’s own body. Their protective
action, however, quickly rejuvenates the body’s

ability to protect against leukemia, so that by 5

days the process is well established.

1729. Factors influencing development of
experimental hypertensive vascular disease
during adrenal regeneration in the rat.
FLtoyp R. SKELTON AND JAMES GUILLEBEAU.*
Dept. of Pathology, Med. College of Georgia,
Augusta.

It has been shown (SKELETON, 1955) that rats
sensitized by uninephrectomy and sodium chlo-
ride administration develop hypertension and
necrotizing vascular disease during the regenera-
tion of adrenal cortical tissue which follows surgi-
cal enucleation of these glands. The present
experiments were performed to determine the
relative importance of the following factors in
the devélépment of this hypertensive vascular
disease: 1) presence of regenerating adrenocorti-
cal tissue, 2) reduction in renal mass and $)
sodium chloride intake. Thus, the development
of hypertension and vascular lesions has been
studied in rats in relation to adrenocortical
regeneration under the following conditions: 1)
after hypophysectomy, 2) with one adrenal intact
and the contralateral adrenal enucleated, 3) with
and without uninephrectomy, 4) with saline and
with water as drinking fluid. Both hypophy-
sectomy and the presence of one intact adrenal
completely inhibited the regeneration of the
adrenal cortex and the development of hyper-

FEDERATION PROCEEDINGS

Volume 16

tensive vascular disease. The presence of both
kidneys and the substitution of water for saline
drinking fluid markedly reduced but did not com-
pletely prevent the development of hypertension
and vascular lesions. Therefore, it is concluded
that of the factors studied, the presence of re-
generating adrenocortical tissue is of most im-
portance in the development of this type of
experimental hypertensive vascular disease and
that reduction of renal mass and excess sodium
chloride consumption simply facilitate the de-
velopment of these changes.

1730. Weight maintenance requirements of
rats fed high protein, fat or carbohydrate
diets. CHARLES A. SLANETZ,* Hans Kaunitz,
Rutu ELLen JoHNson* anv Jacques GUIL-
MAIN.* Inst. of Research in Animal Diseases and
Dept. of Pathology, Columbia Univ., New York
City.

The caloric requirements for weight main-
tenance of weanling rats fed a complete purified
diet decreased from about 2 cal./gm b. wt/wk. to
roughly 1 cal. after 4 wk. and were nearly con-
stant thereafter. This was true with diets con-
taining 10% lard and 30% casein. When diets
containing up to 75% casein or 60% fat and no
carbohydrate were used and when the experi-
ments were carried out for several months, the
caloric requirements were persistently 10%-20%
lower than for animals on high carbohydrate
diets. In calculating the calorie value of the
diets, it was assumed that values of 4 cal./gm for
carbohydrate or protein and 9.2 cal. for fat are
correct. Thus it appeared that carbohydrate
raised the caloric requirements for weight main-
tenance. Replacement of some of the fresh fat by
highly autoxidized fats or some of their fractions
obtained by molecular distillation increased the
caloric requirements for weight maintenance
considerably.

1731. Idiopathic necrosis of bone in labora-
tory mice. LEON SoKoLorr AND Rosert T,
HABERMANN (introduced by J. H. Bragpon).
Natl. Inst. of Arthritis and Metabolic Diseases,
Natl. Insts. of Health, Bethesda, Md.
Circumscribed areas of necrosis of bone have

been observed as a spontaneous lesion in approxi-

mately 4% of laboratory mice during the 2nd yr.
of life. The incidence varied considerably among
the strains studied. The mice were maintained on4
standard diet and, so far as is known, were not
subjected to toxic substances or traumatic han-
dling. The necrosis appears to be of aseptic type.

It simulates some of the idiopathic types of bone

infarcts and aseptic necrosis of man. Minute foei

of necrosis of similar type are found in the ossicles
of the knee in a larger proportion of the animals.

=
awe 1A

ume 16

f both
- saline
yt com-
tension
cluded
of re-
st. im-
ype of
se and
sodium
he de-

nts of
ydrate
.\UNITZ,
GUIL-
ses and
w York

main-
yurified
/wk. to
ly con-
ts con-
n diets
and no
experi-
hs, the
%o- 20%
iydrate
of the
‘gm for
fat are
1ydrate
; main-
fat by
actions
sed the
enance

abora-
ERT T,
.GDON),
iseases,

e have
pproxi-
2nd yr.
among
ed on &
ere not
ic han-
ic type.
of bone
ute foci
ossicles
nimals.

March 1956

There is -a predilection for the epiphyseal and
metaphyseal portions of the long bones to be
affected. The femur is involved most often.
Limited lesions may heal with fibrosis and new
bone formation. In some instances, fracture of the
neck of the femur and collapse of epiphyses occur.
A similar lesion is observed on occasion in the
epiphysis of thoracolumbar vertebrae in kyphotic
rats, and has been seen once in a Syrian hamster.
The etiology of the lesion is obscure.

1732. Endocrine background of chronic cystic
mastitis and adenofibrosis. SHELDON C.
SoMMERS AND ANDRE MarrTeE..* Massachusetts
Memorial Hosp., Boston.

Twenty cases each of women with chronic cystic
mastitis, with adenofibrosis or negative mammary
tissue diagnosed microscopically at autopsy were
investigated for morphologic changes in the
endocrine glands and target organs. Ovarian
stromal hyperplasia occurred respectively in 19,
17 and 1 of the 3 groups, and a proportional stromal
thecomatosis was usually present. Clear-cut
endometrial hyperplasia was found in a majority
of the women with cystic mastitis, in about } of
adenofibrosis cases and in + of the negative control
series. Thyroid glandular abnormalities were
about twice as common accompanying cystic
mastitis as in the other 2 groups. Pituitary cyto-
logic alterations were correlated with the other
morphologic endocrine abnormalities observed in
these women with benign fibrocystic breast
lesions.

1733. Eclampsia in rats produced by con-
trolled diet. F. W. SramierR (introduced by
E. D. Warner) Dept. of Pathology, Univ. of
Iowa, Iowa City.

A diet containing 61% corn starch, 20% casein,
10% brewers’ yeast, 4% HMW salt mixture, and
5% cod-liver oil caused death of a high percentage
of primigravid rats at term when substituted for
the stock (Rockland) ration beginning the 13th
day of pregnancy. Death was preceded by rapidly
progressive cardiorespiratory failure culminating
in violent convulsions. Autopsy findings included
visceral engorgement, extensive pulmonary edema
and massive pleural effusion. Diffuse pulmonary
hemorrhage was commonly present, with oc-
casional renal or adrenal hemorrhage, but intra-
uterine bleeding was usually absent or minimal.
Fibrinthrombi in smaller radicles of the pulmonary
arteries and fibrinoid material occluding renal
glomerular capillaries and arterioles were con-
sistent microscopic findings. Renal parenchymal
disease varied from early tubular degeneration to
eomplete bilateral cortical necrosis. No lesions of
brain or liver were found. Beginning the diet at
the onset of pregnancy caused a high incidence of

-—_
ave 1A

AMERICAN SOCIETY FOR EXPERIMENTAL PATHOLOGY

533

fetal resorption, with correspondingly lower
maternal death rate. Multigravid females were
less subject to eclampsia than primigravid ones,
but susceptibility was increased by starting the
diet earlier in pregnancy or before mating. Cod-
liver oil was found to be the critical component of
the toxic diet. Crude linoleic acid was approxi-
mately as effective but substitution of corn oil
rendered the diet nontoxic. The eclamptic disease
was readily prevented by prior treatment of the
subject animals with alpha-tocopherol or lettuce,
or by adding either of these materials to the
eclamptogenic diet.

1734. Morphologic alterations and dissecting
aneurysms of aorta in cortisone-treated
hamsters. C. HAROLD STEFFEE AND KATHARINE
C. Snetu.* Lab. of Pathology, Natl. Cancer Inst.,
Natl. Insts. of Health, Bethesda, Md.

During experiments in which hamsters were
treated with cortisone acetate in order to accom-
plish heterologous transplantation of human fetal
tissue, 79 of 120 animals died with massive intra-
thoracic or intraperitoneal hemorrhage. Histologic
examinations of the aortas of 50 of these hamsters
revealed dissecting aneurysm in 40, and necrotiz-
ing arteritis with rupture of the aorta in 10. Com-
parable results were obtained in hamsters treated
with cortisone but not bearing transplants of
human fetal tissue. No differences were observed
between animals purchased from a dealer and
those obtained from the colony maintained at the
Natl. Insts. of Health. No aneurysms occurred in
hamsters without cortisone treatment. Histo-
logically, the aneurysmal dissection of the aortic
wall usually appeared just above the aortic valve.
The extravasated blood was found in the region of
the junction of the media and adventitia. Sections
strained with new fuchsin-orcinol, orcein and
Verhoeff’s techniques showed no pathologic change
in the elastic fibers. Inconstant alterations in the
muscle cells consisted of vacuolization of the
cytoplasm, a variable metachromasia with tolui-
dine blue and focal deposition of an amorphous
material which was purplish-red in the periodic
acid-Schiff preparations.

1735. Natural history of aortic atherosclero-
sis, ages 1 to 40. Jack P. Strone,* Henry C.
McGI11, Jr., Oscar R. GrirFin* AND RussELL
L. Houtman. Depts. of Pathology, Louisiana
State Univ. School of Medicine, and Charity
Hosp. of Louisiana, New Orleans.

Studies on the natural history of aortic athero-
sclerosis, with particular emphasis on the early
and possibly reversible lesions, have been extended
to include a total of 304 cases from 1 to 40 yr. of
age. In these cases the percentage of intimal surface
of the aorta involved by sudanophilic ather-

534

omatous lesions and by hyaline or pearly plaques
has been estimated quantitatively both for the
entire organ and for specific anatomic regions.
Analysis of these figures leads to the conclusion
that 1) the rise in severity of involvement with
atheroma for both Negro and white races is sharp-
est between the ages of 5 and 15 yr., but there-
after the lesions remain remarkably constant or
regress slightly; 2) the Negro is more severely
involved with atheroma than the white until age
15 yr., but after 20 yr. the races are approximately
equally affected; 3) the percentage of intimal
surface involved with pearly plaques does not
begin to rise significantly until age 30 yr., 20 hr.
after the rise in atheroma; 4) previously reported
impressions that the thoracic and abdominal por-
tions of the aorta are more severely involved by
atheroma are strongly confirmed; 5) the abdominal
aorta is the site most severely involved by pearly
plaques. Instances of the complications in the
lesions of atherosclerosis which ordinarily lead to
disease—hemorrhage, ulceration, thrombosis—
were practically absent in this group. If our con-
cept that atheromatosis leads to the development
of pearly plaques is correct, it appears that many
years of atheromatosis are necessary before pearly
plaques are formed.

1736. Turnover rate of RNA in rat liver.
Rosert W. Swick aND ARTHUR L. Kocu (intro-
duced by Hermann Lisco). Argonne Nail.
Lab., Lemont, Ill.

Several difficulties encountered in the interpre-
tation of single-exposure tracer experiments at-
temping to measure turnover rates may in part be
eliminated or circumvented by use of the continu-
ous-exposure technique. This technique was used
to measure the renewal rate of RNA in rat liver
using both C'* and P*2. Rats were fed at hourly
intervals a purified diet containing either CaC™“O;
or NasHP#O, for periods up to 16 days. The
specific activity of the precursor pools, in the case
of C, could be inferred with confidence from a
knowledge of the specific activity of intracellular
CO, represented by urea and, in the case of P*,
was found to be equal to the measured specific ac-
tivity of acid-soluble phosphorus in experiments
greater than 16 hr. duration. Hence, the changes
in specific activity of the RNA components can be
used to calculate the parameters of nucleic acid
metabolism. The rate of renewal of rat liver RNA
phosphorus was found to be 17%-21%/day; that
of the RNA purines was 20%/day for adenine and
24%/day for guanine. It therefore appears clear
that the values obtained with C™ are comparable
to those given by P*. It may be concluded that
with either isotope the results obtained give a
valid measure of nucleic acid turnover and that the
molecule is replaced as an entity. Similar conclu-

FEDERATION PROCEEDINGS

Volume |,

sions are drawn from the rate of incorporation @
these isotopes into DNA. (Work performed unde
the auspices of the Atomic Energy Commission.)

1737. Hepatic cholesterol synthesis: its de.
pression by dietary cholesterol and subse.
quent recovery on a cholesterol-free diet,
C. Bruce Taytor, LAVERNE NeEwson,* Mar
JORIE StuMPE,* GeorGE Cox* anp Ray Ts
muRA.* Rush Lab. of Pathology, Presbyterian,
Hosp., Chicago, Ill.

Few data are available concerning the response
of hepatic cholesterol synthesis to cholesterol de.
privation following cholesterol ingestion. Such dats
appear essential to proper interpretation of cho-
lesterol synthesis rates measured in liver biopsies
from patients subjected preoperatively to periods
of cholesterol-free diet (Circulation 12: 489, 1956),
Three groups of rats received 2% cholesterol-5%
corn oil diet: group I for 1 day; group IT, 1 wk.
group III, 2 months. Animals from each group
then received cholesterol-free rations for varying
periods, and hepatic cholesterologenesis He
radioacetate was determined by in vitro incubation
with 1-C™ sodium acetate. Hepatic cholesterolo-
gensis in control animals was 0.6-3.0 mg/100 gm
liver/hr.; av., 1.65 mg. Cholesterol feeding for]
day, wk. and 2 months produced maximal and
equal depression of synthesis (rates: 0.01-0.08).
Group I recovered near normal synthesis after |
day of cholesterol derpivation (rates: 0.8-1.5; av,,
1.07). Group IT showed minimal recovery of syn-

thesis in 2 days, appreciable after 4 days (rates:
0.5-0.7; av., 0.63) and near normal values after 6
and 8 days. Group IIT showed partial recovery a
4 days (rates: 0.189-0.86; av., 0.52) and 9 days,
with near complete recovery in 15 days (rates:
1.0-1.8; av., 1.5). Older animals had lower control
synthesis rates and showed slower recovery than
young animals. (Supported by Chicago and
American Heart Assocs., Life Insurance Med.
Research Fund and Otho S.A. Sprague Memorial
Inst.)

1738. In vitro catabolism of protein hormone
by liver: effect of other proteins on the gly-
cogenolytic action of glucagon. H. #.
Tomizawa,* J. M. Typercuern,* Y. D. Har
sEY* anD R. H. Wiuurams. Dept. of Medicine,
Univ. of Washington, Seattle.

Experiments in this laboratory concerning it-
activation of insulin suggest that. a-cortieotropin,
casein, glucagon and growth hormone, as well #&
insulin, can be degraded by an enzyme system of
liver. Conversely, bovine plasma albumin a-lactal:
bumin and ribonuclease are probably not sub;
strates (J. Biol. Chem. 217: 685, 1955). Work con}
cerning this question of substrate specificity was
extended by determining the effect of the above

Za 2 A

ce]
we
all
spe
the
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ee diet,
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tay Ta
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response
terol de-
uch data!
. of cho-
biopsies
) periods
9, 1956).
terol-5%
rT, 1 wk.
h group
varying
is from
cubation
esterolo-
/100 gm
ng for!
mal and
01-0.08).
s after!
-1.5; av,
, of syn-
s (rates:

s afteré
overy at
| 9 days,
s (rates:
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ery than
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icity was
1e above

March 1956

mentioned protein on the glycogenolytic action of
glycagon: Under the conditions used, 0.15 yg of
glucagon was too small a quantity to produce a
significant glycogenolytic effect in rabbit liver
slices. However, the addition of insulin, a-cortico-
tropin, a-casein or growth hormone to this quan-
tity of glucagon resulted in glycogenolytic effects
only possible when larger quantities of glucagon
alone were used. Bovine plasma albumin, a@-lactal-
bumin and ribonuclease were not effective in this
manner. Since these observations are strikingly
similar to those obtained in studies on the in-
activation of insulin, they suggest that glucagon
was partially spared from degradation by the
successful competition of other substrates. These
results support the hypothesis that insulin,
a-corticotropin, a-casein, glucagon and growth
hormone can be degraded by the same enzyme
system of liver. A considerable purification of the
degradative activity of beef liver towards insulin
has been effected.

1739. Use of fresh and frozen adult and em-
bryonic skin homografts in rabbit and man.
HELENE Wa.uuace Toouan (introduced by
CornELIUS P. Ruoaps). Surgical Research Unit,
Brooke Army Hosp., Fort Sam Houston, Texas.
Studies on 400 rabbits properly conditioned with

cortisone and allied steroids, a technique previ-
ously employed by the author for the successful
transplantation of human tumors and embryonic
tissues in laboratory animals (Cancer Research 14:
660, 1954; Proc. Soc. Exper. Biol. & Med. 86: 607,
1954) have shown that homgrafts of adult or
embryonic skin will ‘take’ as well as autografts
and remain in excellent condition as long as the
treatment continues (periods of 4 months and
more). Correct dosage is all important for good
results. Both fresh and frozen tissues were used
successfully for grafts. The grafts remain intact
for approximately 1 month after steroids are dis-
continued and are then sloughed, with a few ex-
ceptions. The preliminary results on the practical
use of human embryo skin for burn patients are
encouraging.

1740. Methyl group synthesis in leukemia-
susceptible mice fed a folic acid-deficient
diet and in leukemic mice fed a normal diet.
M. Toporek* anp F. H. BETHELL. Simpson
Memorial Inst., Univ. of Michigan, Ann Arbor.
Otherwise normal male mice of a leukemia-sus-

ceptible strain fed a diet deficient in folic acid

were found to produce liver choline from parenter-
ally administered sodium formate-C™ with lower
specific activity than did male controls even
though the liver folic acid level was only slightly,
if at all, reduced below the normal level. The basic
diet and experimental conditions were reported

AMERICAN SOCIETY FOR EXPERIMENTAL PATHOLOGY

535

previously (Abstracts, 128th Natl. Meeting, Am.
Chem. Soc., p. 7C, Sept. 1955). Female mice on the
deficient diet did have a definitely lowered liver
folic acid level but managed to synthesize choline
with as high specific activity as the normal female
controls. This observation of a sex difference is
similar to that reported previously with respect to
a dietary deficiency of Bi2. Mice of the same strain,
both male and female, in which leukemia has been
induced by transplantation, synthesize liver
choline with definitely higher specific activity
than do the normal controls. These mice were fed
a normal diet (Rockland Mouse Diet). The period
between transplantation of the leukemia and ex-
periment was 7 days. Liver B,. levels were possibly
up slightly in males and down in females. Citro-
vorum factor levels were definitely down in 1
female and 1 male group and stable in 1 female and
1 male group. As yet, the data show no definite
correlation between metabolic efficiency in the
synthesis of labile methyl groups and liver stores
of B,2 and folie acid. (Supported by Public Health
Service Grant C-1994-C2.)

1741. Microscopic study of integrated pat-
terns of vascular and extravascular disease
due to hypervitaminosis D. RicHarp TRUE-
HEART,* MARJORIE STUMPE,* GEORGE Hass AND
C. Bruce Taytor. Rush Lab. of Pathology, Pres-
bytertan Hosp., Chicago, Ill.

Rabbits given irradiated ergosterol developed a
generalized disease characterized principally by
resorption of bone and abnormal extraosseous dep-
osition of calcium. Each part of the circulatory
system and most tissues had a characteristic sus-
ceptibility or resistance to the inflammatory-
degenerative-calcific sequences of the disease. On
moderate dosage, changes which appeared first in
the inner media of the aortic arch soon spread in
depth and then distally along the aorta and into
the major branches. With 2 exceptions, the disease
usually decreased in severity in successively
smaller branches. The first exception occurred
with high dosage. These animals developed a
panarteritis restricted to and augmenting de-
generative-calcific disease of intrinsic arteries of
cardiac and skeletal muscle. The second exception
occurred in special arterial systems where changes
increased in severity in successively smaller
branches. This was conspicuous in the spleen,
kidney, salivary glands, duodenum and _ acid-
secreting division of the stomach. Patterns of
vascular disease were no. less complex than ‘pat-
terns of degenerative-calcific changes in extra-
vascular tissues, but it was clear that vascular
changes usually preceded extravascular changes in
integrated functional systems and that extra-
vascular changes never occurred in tissues where
arterial systems were always free from disease.

536

These relations, together with the great suscepti-
bility of certain arterial systems and absolute
resistance of others to the inflammatory-degener-
ative-calcific changes, indicate that experimental
hypervitaminosis D should be a useful source for
ideas concerning pathogenesis of human arteri-
osclerosis and associated disturbances of ad-
vancing age.

1742. Inhibition of myeloid leukemia induc-
tion in RF mice by partial shielding during
irradiation. ARTHUR C. Upton, JacosB FurtH
AND Escuo. 8S. LEprorp. Biology Div., Oak Ridge
Natl. Lab., Oak Ridge, Tenn., and Children’s
Cancer Research Fndn., Boston, Mass.

Male mice of the RF strain, 5-6 wk. of age, were
randomly divided into treatment groups of 75-100
animals and subjected to 1) whole-body 250-kvp
x-irradiation (150, 300 or 450 r), 2) partial-body
irradiation (450 r) or 3) no treatment (controls).
Partial-body irradiation was performed by shield-
ing the lower extremities and pelvis with 11 mm of
lead, which reduced the dose to approximately 2.5%
of that administered to the unshielded tissue; the
mass of tissue shielded was estimated to constitute
less than 4 the entire body. The incidence of
myeloid leukemia observed in the various treat-
ment groups was as follows: controls (Or), 1%; 140r,
26%; 300 r, 44%; 450 r (total-body), 22% and 450 r
(partial-body), 8%. The incidence of thymic
lymphomas in the same groups was: controls (0 r),
2%; 150 r, 9%; 300 r, 19%; 450 r (total-body), 28%;
and 450 r (partial-body), 7%. As observed previ-
ously (Cancer Research 14: 682, 1954), the RF
mouse appeared more susceptible to the induction
of myeloid leukemia than to the production of
thymic lymphomas, the incidence of the former
being greatly increased by a single dose of only
150 r. Although it has been reported by others that
lymphoma-induction may be inhibited by shield-
ing hemopoietic tissue (or by the injection of bone
marrow cells postirradiation), the present experi-
ments demonstrate that for the same integral dose
of radiation the induction of myeloid, as well as
lymphoid, leukemia was greatly reduced by par-
tial-body shielding.

1743. Fibrinogen polymerization test. BRUNO
W. VoLK anp SamuEt Losner.* Isaac Albert
Research Inst. of the Jewish Chronic Disease
Hosp., Brooklyn, N. Y.

We have demonstrated previously that the
transition of fibrinogen into fibrin is rendered in-
complete by minimal amounts of heparin. Although
coagulation is considerably delayed a solid clot is
formed. Subsequently centrifugation separates
the coagulum from serum which is extraordinarily
rich in fibrinogen. We have attributed this phe-
nomenon of fibrinogen preservation in serum to
the interference with fibrinogen polymerization

FEDERATION PROCEEDINGS

Volume 16

by heparin. Utilizing critical doses of heparin and
blood we have elaborated a procedure designated
by us as the ‘fibrinogen polymerization test’ which
is based upon the phenomenon of fibrinogen pres-
ervation in serum. The presence of fibrinogen in
the supernatant serum 2 hr. after gross coagulation
has thus been commonly observed in the blood of
healthy individuals. This result was considered
‘negative.’ On the other hand, absence of fi-
brinogen 2 hr. after gross coagulation, called a
‘positive’ test, has been observed in active rheu-
matoid arthritis, acute rheumatic fever with
carditis, chorea minor, nonspecific myocarditis
and pericarditis, and also during the 2nd, 3rd and
4th wk. of acute glomerulonephritis. In order to
obtain experimental confirmation of accelerated
fibrinogen polymerization, this procedure was
applied to a series of rabbits in whom Shwartz-
man’s phenomenon had been produced. While the
fibrinogen polymerization test was found to be
uniformly negative during the Ist wk. after ap-
pearance of the hemorrhagic necrosis, it became
positive from the 8th-20th day thereafter. In con-
trast, the great majority of normal rabbits dis-
played negative fibrinogen polymerization tests.

1744. Experimental carcinoma of the cervix.
E. von Haam anv D. G. ScarpE.ui.* Dept. of
Pathology, College of Medicine, Ohio State Univ.,
Columbus.

In previous experiments (Cancer Research 15:
449, 1955) the authors studied the pathogenesis of
3,4-benzyprene-induced carcinoma of the cervix
and vagina in C3H mice by comparative cytologic
and histologic methods. In the present series of
experiments the application of the 3,4-benzpyrene
was stopped at various stages of the development
of the experimental carcinoma of the cervix in
C3H mice, as evidenced by periodic studies of the
vaginal smear. Prompt regression of the inflam-
matory cells was noted and, if the carcinogen was
discontinued at an early stage of the experiment, a
complete recovery of the animals could be ob-
served. If the carcinogen was discontinued after
the appearance of atypical cells in the vaginal
smear of the animals, a large percentage of the
mice developed invasive carcinoma, although it
usually appeared later than in the control group,
in which the painting with 3,4-benzpyrene was
continued. Quantitative microspectrophotometri¢
measurements of the DNA content of the nuclei of
exfoliated cells throughout the experiment were
undertaken in order to discover any significant
cytochemical changes in the exfoliated cells of the
animals which recovered after discontinuation of
the carcinogen.

1745. Enzymatic staining reactions in the
kidney following interruption of renal cir-
culation. M. WacusteIn anp E. MBIsEL.*

his
sta

mel
The

ume 16

in and
mated
which
| pres-
yen in
lation
ood of
idered
of fi-
lled a
rheu-
with
irditis
‘d and
der to
erated
> was
wartz-
ile the
to be
er ap-
ecame
n con-
s dis-
ests.

ervix.
opt. of
Univ.,

ch 15:
esis of
cervix
ologic
‘ies of
yyrene
pment
vix in
of the
nflam-
n was
ent, &
ye ob-
after
aginal
of the
ugh it
group,
e was
metri¢
clei of
L were
ificant
of the
ion of

1 the
il cir-
ISEL.*

March 1956

Dept. of Pathology, St. Catherine’s Hosp., Brook-

lyn, N.Y?

Of the many enzymes known to be present in the
mammalian kidney, some can be demonstrated by
histochemical techniques. These reveal consistent
staining patterns in a given species (WACHSETIN.
J. Histochem. 3: 246, 1955). The effects of experi-
mentally induced anoxia were studied in the rat.
The renal pedicle of the left kidney was ligated
under Nembutal anesthesia and the perinephric
fat stripped from the renal capsule. The right
kidney served as control. The animals were killed
after 1, 2, 3, 4, 6, 8, 12, 24 and 48 hr. 8 hr. Frozen
sections were cut at 10 w from fresh and neutral
formalin-fixed material and studied with the
following techniques: succinic dehydrogenase,
DPN diaphorase, glucuronidase, esterase, non-
specific alkaline and acid phosphatase, nonspecific
phosphatase at a pH of 7.2, 5-nucleotidase, ade-
nosine triphosphate and glucose-6-phosphatase.
In addition, the plasmal reaction in fresh frozen
sections and the PAS stain in fixed material were
used. Considerable differences with various en-
zymatic staining techniques were found. Changes
were first noticed in sections stained for DPN
diaphorase and glucuronidase, but not earlier
than 3-4 hr. after interruption of the renal circu-
lation. Among the phosphatases, glucose-6-phos-
phatase and, to a lesser degree, 5-nucleotidase
proved to be most sensitive. The findings will be
compared with those in localized renal damage
induced by nephrotoxic substances. (Aided by
grant A-688 of the Public Health Service.)

1746. Selective adsorption of antihemophilic
factor (AHF) and fibrinogen from canine
plasma. Ropert H. Waener,* J. G. McALuis-
TER III* anv K. M. Brinxuovs. Dept. of Pathol-
ogy, Univ. of North Carolina, Chapel Hill.

A number of materials were screened for po-
tential usefulness as selective adsorbents for
fibrinogen and antihemophilic factor (AHF).
Fibrinogen and AHF were found to be adsorbed by
many substances, but percentage recovery on
elution was very low. AHF was largely lost from
Plasma on passage through glass or asbestos
sterilizing filters. Aluminum hydroxide, in con-
trast to barium sulfate, adsorbed appreciable
amounts of AHF from oxalated plasma. Proper
amounts of several materials, including fuller’s
earth, Florigel and some grades of aluminum oxide
were found to adsorb fibrinogen without adsorp-
tion of AHF, Elution of fibrinogen was not success-
ful. Preliminary fibrinogen adsorption by Florigel,
followed by cold ethanol fractionation, provided
the most successful method for purifying and con-
centrating canine AHF. This procedure was also
wed with bovine plasma. Bidwell’s technique of
salting out AHF from bovine plasma with phos-
phate buffers was also studied, with the inclusion

AMERICAN SOCIETY FOR EXPERIMENTAL PATHOLOGY

537

of a preliminary fibrinogen adsorption step. The
kaolin adsorption-elution procedure, using canine
plasma, did not yield a potent AHF fraction. A
series of bovine and canine AHF fractions pre-
pared by adsorption and precipitation were as-
sayed for purity and then tested by parenteral
injection into hemophilic dogs. (Aided by a grant
from the Natl. Heart Inst., H-1648, Natl. Insts.
of Health, PHS.)

1747. Importance of burn area vs. burn depth
in systemic responses to experimental
flash burns. MACKENZIE WALSER AND LEONARD
J. BopENLos (introduced by G. BRECHER).
Naval Med. Research Inst., Bethesda, Md.

A source of intense thermal radiation (7.5
Cal/cm?-sec.) consisting of a battery of flood
lamps was used to produce extensive flash burns
in depilated rats. By rotating the animals in a
special device, it was possible to expose up to 35%
of the body surface to brief thermal ‘pulses.’ One
group of animals (A) was subjected to a burn of
35% body surface by a 0.75-sec. ‘pulse’ and another
group (B) received 14% burns from a 1.9 sec.
‘pulse.’ Thus both groups received approximately
equal ‘doses’ ot radiant energy. No therapy was
given. Five to 10 rats were killed at 3, 9 and 24
hr. Pronounced hyponatremia occurred in group
B but none in group A. NPN rose from 28 mg% to
70 mg% in both groups, though the rise was de-
layed until 9 hours in group B. Plasma volume
determined by T-1824 fell 32% in group A and
42% in group B, within 3 hr.; at 24 hr. it had re-
turned to normal in group A but remained low in
group B. These changes are statistically signifi-
cant. No significant alterations in serum chloride,
potassium or total protein concentrations oc-
curred in the burned animals. Control rats showed
only a slight fall in plasma volume, due to depila-
tion and anesthesia. The systemic response to
extensive flash burns in rats includes hyponatremia
hypovolemia and azotemia, with no change in
serum chloride, potassium or protein concentra-
tions. Within the limits of the experimental con-
ditions used, the response correlated with burn
depth rather than area.

1748. Comparative pathology of androblas-
toma. Nancy WARNER,* NATHAN B. FRIEDMAN
AND Francis Masin.* Div. of Labs., Cedars of
Lebanon Hosp. and Dept. of Pathology, Univ. of
Southern California, Los Angeles.

Destruction of the left ovary in fowl is known
to result in certain changes suggesting masculinza-
tion. The tumor-like proliferation of the right
gonad which results in these animals resembles
certain masculinizing ovarian tumors in women
which have been described as arrhenoblastoma or
androblastoma. The hormonal excretion of these
birds was studied and comparisons made with the

538 FEDERATION

changes found in the ovaries of aged rats and in
the gonads of rats transplanted to the spleen and
thymus of castrate animals. (Supported by Public
Health Service Grant C 1789.)

1749. Biochemical studies on beta-irradiated
guinea-pig epidermis. CHARLES WEISS AND
JosepH TasacHnick. Dept. of Microbiology,
Albert Einstein Med. Ctr., Northern Div., Phila-
delphia, Pa.

With the aid of a Sr® applicator, varying doses
of beta radiation were applied locally to the sur-
face of clipped, albino guinea-pig skin. Employing
450-550 gm animals, about 5500 rep resulted in a
slight inflammatory response 24 hr. after irradia-
tion. An intense inflammatory reaction, with
dermonecrosis and permanent hair loss, appeared
about 8-10 days after irradiation. In newborn
guinea pigs, about 2000 rep was sufficient to elicit
this response. Animals weighing 500 gm, which had
received a single dose of 6800 rep over 1 inch square
area of closely clipped skin, were killed at various
time intervals and the epidermis removed and
homogenized. There was no observable change in
epidermal RNase, DNase or phosphatase (pH 7.2)
activities over a 40-day period when compared
with homologous sections of normal epidermis.
Two UV-absorbing compounds, not found in
normal epidermis, were separated by paper
chromatography from the acid soluble fraction of
epidermis removed 8 days after irradiation. As yet,
sufficient amounts have not been isolated for
chemical identification. Sixteen days after irradi-
ation there was an increase in total free amino
acids in the irradiated epidermis which continued
to increase to the 40th day. Jn vitro studies with
depilated excised skin kept at 4°C showed no
change in epidermal nuclease or phosphatase ac-
tivities when 11,300 beta rep were administered.
With 22,000 rep there resulted a 40% decrease in
RNase activity after 10 min. incubation. There
was no inhibition of DNase with as high as 34,000
rep. (Supported by a grant from the Atomic
Energy Commission Contract AT(30-1)-1727.)

1750. Effects of x-radiation and cortisone on
serum proteins in mice. ALVAR A. WERDER,
CREIGHTON A. HarpIN AND PERRY MorGAN
(introduced by Tom R. Hamiuron.) Depts. of
Med. Microbiology and Surgery, Univ. of Kansas
Med. Ctr., Kansas City.

A large number of studies have been made on
the influence of x-radiation and cortisone on anti-
body formation and immunity in animals but
relatively few describe changes in electrophoretic
patterns of serum proteins in such animals. To
investigate the latter in mice, 640 CFW mice,
approximately 6 wk. of age, were subjected to
either 1) total body irradiation (300 r) or 2) injec-

PROCEEDINGS

Volume ij

tion of cortone acetate (4 mg of saline suspension),
After treatment with either radiation or cortisone,
mice in groups of 20 were bled at intervals 4
follows: 18, 24, 48 and 72 hr.; 1, 2 and at time
4 wk. Pooled sera were collected and the serum
proteins were analyzed in a Tiselius electro.
phoretic apparatus. Total protein concentration
(5.6%) remained essentially unchanged. Pattern
of relative concentrations of serum proteins wer
changed as shown in table 1.

TaBLE 1. ALTERED CONCENTRATIONS INDICATING
PERCENTAGE INCREASE (+) OR DECREASE (—)

Alb.

24 hr. X-ray +5
cort. —3

1 wk. X-ray +4
cort. 0

2 wk. X-ray +11
cort. +1

1 mo. cort. —14

a-glob.

—16

+9
—26
+29
+18

+4
+48

B-glob.

+15
—14
+57
—28
—22

+1
—34

y-glob,
—19
—§
—67
—67
+40
—14
—40

It is of special interest that in cortisone-injected
mice the concentration of gamma globulin r.
mained below normal for as long as 1 month while
the alpha globulin concentration was consistently

above normal.

1751. Effect of fractionation of dose of «x:
radiation upon recovery of the rat small
bowel epithelium. R. BLanp WILLIAMs, JR.
Jutius Wuite* ANp JANE N. Toau.* Naval Med.
Research Inst., and Natl. Cancer Inst., Natl.
Insts. of Health, Bethesda, Md.
Recovery of cell division was studied in the rat

small bowel epithelium following single doses of

x-rays from near threshold to tissue lethal doses.

Then the total dose was fractionated to permit

an evaluation of 1) recovery of the tissue prior to

repeating the initial dose and 2) the effect of keep-
ing the total time and total dose constant, but

varying the magnitude of

individual

doses.

Divided doses were not as effective as single doses
in terms of delay in recovery (prolongation of
interphase) or destruction of crypts. A second dose
of 600 r, 96 hr. after the first merely repeated the
delay in division and recovery cycle. If this dose
was given 12 hr. after the initial irradiation, severe
destruction of large numbers of crypts and differ.
entiation of their lining stem cells occurred. Nine
hundred roentgens was given over a period of 12
hr. as follows: 225 r every 4 hr.; 450 r, followed
12 hr. later by 450 r; 700 r followed 12 hr. later by
200 r. The degree of recovery was dependent upo
the size or intensity of individual doses. Recovery
was greatest following 4 doses of 225 r, and least
after 700 r followed 12 hr. later by 200 r. The rate
of DNA synthesis following such doses supported
and reflected changes in the rate of cell division.

‘olume Ij

pension),
ortisone,
ervals a
at time
he serum

electro.
ntrations
Pattern
eins were

DICATING
SE (—}

b. —-y-glob,
—19
-5
—67
—67
+40
—i4
—40

ee Ww ON eS OT

Injected
bulin re-
nth while
isistently

se of x
at small
AMS, JR.,
aval Mei.
st., Nall.

in the rat
. doses of
hal doses.
to permit
e prior to
t of keep-
tant, but
al doses.
ngle doses
gation of
cond dose
eated the
this dose
on, severe
ind differ-
‘red. Nine
riod of 12
, followed
r. later by
dent upon
Recovery
and least
. The rate
supported
| division.

March 1956

Doses na longer appreciably additive in their
effect upon the small bowel epithelium were still
cumulative upon the bone marrow.

1752. Inhibition of elastase. KATHLEEN K.
WINTER AND SAM FRANKEL (introduced by H.
T. BLUMENTHAL). Section of Labs., Jewish Hosp.
Research Fndn., St. Louis, Mo.

Using the colorimetric method of Sachar eé al.
(Proc. Soc. Exper. Biol. & Med. 90: 323, 1955),
some studies were done on the cation and anion
inhibition of the pancreatic enzyme elastase
which has been implicated in the degeneration of
elastic tissue. The enzyme was found to be very
sensitive to the ionic strength of its medium. The
addition of NaCl to the physiological concentra-
tion caused approximately 50% inhibition,
whereas a final molar concentration of .01-.02
gave a slight increase in activity when compared
with a H.O control. Similar results were obtained
with KCl, MgCl, CaCl, Na2SOu, Nal, etc.,
thereby showing this inhibition to be nonspecific
with regard to the ion involved and dependent
merely on total ionic strength. The metal salts of
Hg, Zn, etc., even though many of them formed
insoluble hydroxides at the alkaline pH used, were
markedly inhibitory at the molar concentration
employed. Preliminary studies on the serum in-
hibition of elastase showed remarkable similarity
to the serum inhibition of trypsin. In conditions
where the antiproteolytic titer was elevated the
antielastase titer was also high, suggesting either
that this function of serum is nonspecific in its
ability to inhibit certain enzymes or that serum
contains a separate antielastase inhibitor which
varies along with the antiproteolytic fraction.
Dialysis of the serum to remove the salts did not
nullify its ability to inhibit elastase.

1753. Role of arterial and renal injury in
production of atheromatous lesions in
coronary arteries of the rat under various
dietary conditions. Ropert W. WIssLER,
Rosert F. AuLiLeN,* Ricnarp H. Moy* anp
WiuuraM L. Braprorp.* Dept. of Pathology, Univ.
of Chicago, Chicago, Til.

Acute experiments with adult male rats fed
tations high in animal lipid have confirmed the
coronary atherogenic effect of the combined ad-
Ministration of antikidney serum (AKS), desoxy-
corticosterone acetate (DCA) and sodium chloride
(NaCl). No acute lesions have been observed when
either the DCA or AKS was omitted even though
hypercholesterolemia of approximately com-
parable degree was observed when only AKS and
NaCl were administered. The levels of dietary
protein and lipid were varied over a wide range in
adult male rats receiving AKS followed by DCA
and added NaCl. Coronary atheromatous lesions

AMERICAN SOCIETY FOR EXPERIMENTAL PATHOLOGY

539

and marked elevations in serum cholesterol and
lipid phosphorus concentrations were observed in
animals receiving either a high fat, high choline,
low protein ration or a low fat, low choline, high
protein ration. A low protein, low fat, high choline
ration administered to rats receiving the combined
AKS, DCA, NaCl treatment resulted in less eleva-
tion of the serum cholesterol and lipid phosphorus
concentrations and no lesions were observed in
the coronary arteries. A ration high in lipid, pro-
tein and choline produced an even more severe
elevation in the serum cholesterol without a com-
parable rise in lipid phosphorus when this dietary
treatment was combined with the disturbance in
lipid metabolism resulting from AKS, DCA and
NaCl treatment. This resulted in a marked rise in
the SC/LP ratio and a high incidence of coronary
atheromatous lesions. These results were con-
trasted with the effects of variations of these
same diet ingredients upon the SC/LP ratio in
animals receiving dietary treatment alone. (Sup-
ported by Public Health Service H2174 and A374.)

1754. Hemoglobin and derivatives as antibac-
terial factors in serum. Don C. Woop Anp
JoE Ono (introduced by Russetu S. JoNnsgs).
Fort Douglas VA Hosp., and Dept. of Pathology,
Univ. of Utah College of Medicine, Salt Lake
City.

While studying the effects of specific antibodies
on the rate of bacterial growth, it was observed
that excess antiserum caused a marked inhibition
not dependent upon the antibody. Using oxygen
uptake in a Warburg apparatus as a criterion for
growth, at least 2 systems appear to be associated
with the bactericidal activity of serum: a) the
properdin system which requires complement and
is labile at 56°C, b) a second system, apparently
independent of complement, is stable at 56°C,
but destroyed at 75°C. Because serum from blood
which had very slight hemolysis always resulted
in greater bactericidal activity than serum with-
out visible hemolysis, the antibacterial activity
of hemoglobin and other porphyrin-containing
molecules was studied. These compounds were
suspected of being associated with the second
system which inhibits bacterial growth. The
influence of the following solutions on the growth
of FE. coli and B. subtilis were studied: serum,
laked erythrocytes, crystalline hemoglobin, crys-
talline hemin, acid hematin, alkaline hematin,
crystalline catalase, cytochrome c, chlorophyll,
bilirubin and biliverdin. Hemoglobin showed the
greatest inhibitory activity; marked inhibition
in the growth of bacteria was measured in those
Warburg flasks containing microgram quantities
of hemoglobins. Thus, minute traces of hemo-
globin account for a part of the antibacterial
activity reported for serum.

540

1755. Induction by sulfathiazole of alterna-
tive pathways for biosynthesis of nucleic
acid purines in Escherichia coli. RoBert C.
Woop* anp M. G. Seva (introduced by Stuart
Mupp). Dept. of Microbiology, Univ. of Penn-
sylvania School of Medicine, Philadelphia.
Sulfathiazole has been found to exercise an

unequal effect on the incorporation of glucose-C™

into PNA adenine and guanine in 2 strains of Z.

coli. In wild strain B, grown in a basal medium of

casein hydrolysate and glucose-C"™, sulfathiazole
produces a 40% depression in the incorporation of
glucose-C™ into adenine. On the contrary, sulfa-
thiazole increases the incorporation of glucose-C"
into guanine in strain B. When a p-aminobenzoic
acid-requiring mutant (M48-34), which accumu-
lates 5-amino-4-imidazolecarboxamide (AICA) in
the absence of sulfathiazole, is grown in a basal
medium containing salts, glycine, glutamic acid,
p-aminobenzoic acid and glucose-C, the con-
tribution of glucose-C" to adenine and to AICA is
not inhibited by sulfathiazole. However, sulfa-
thiazole causes a 32% inhibition of the incorpora-
tion of glucose-C™ into guanine in this mutant.

Assuming that a reduction in the relative specific

activity obtained for the incorporation of glucose-

C'™ may mean that another carbon source must

compensate for the reduction, then sulfathiazole

‘induces’ the biosynthesis of a portion of adenine

in strain B and of a portion of guanine in strain

M48-34 by a pathway(s) not involving glucose.

Moreover, since sulfathiazole does not inhibit the

contribution of glucose-C'* to AICA in strain

M48-34, it appears that the alternative pathway (s)

for the biosynthesis of guanine may not proceed

via AICA. The role of these alternative pathways
for purine biosynthesis in relation to resistance to
sulfathiazole was also studied.

1756. Etiologic studies of febrile respiratory
disease in navy recruits with emphasis on
APC viruses. R. L. Wootripce, J. T. Gray-
ston, J. E. WuitesipE anp M. FRIEDMAN.
(introduced by C. G. Loost1). Naval Med. Re-
search Unit number 4, Great Lakes, and Dept.
of Medicine, Univ. of Chicago, Chicago.
Etiologic studies were performed on Naval

recruits with febrile respiratory illnesses from

September, 1954 through August, 1955. Respira-

tory diseases were low (4/1000/wk.) during the

fall of 1954 but rose to a peak (48/1000/wk.) in

February at which time streptococcal infections

were prevalent. A few cases of influenza B occurred

in January. The APC group of viruses, however,

FEDERATION PROCEEDINGS

Volume 1§

were present in the Recruit Training Comman(
during the entire year. Complement fixation tests
with APC type 4 (RI-67) antigen were performed
on 1160 paired serums, and 4-fold antibody rises
were found in 456. During the fall months legs
than 10% of the serums had an antibody rise,
Throughout the rest of the year at least 30% of
the serums were positive in each month with
peaks of 60% in February, March and July. Sixty
APC viruses were isolated from 288 nasal washings
in HeLa cell cultures. Typing these viruses re-
vealed four type 3 and one type 6 in September
and October. 7'ype 7 predominated during January
and February with 26 isolated. In addition one
type 3 (Jan.) and one type 4 (Feb.) were found.
Except for two type 7 viruses in March the re-
maining 25 viruses isolated were type 4. Of interest
was the continuing high isolation rate of type 4
virus during the spring and summer months.

1757. Pathological changes in dogs and rats
from feeding of coumarin. RoBErt E. Zwic-
KEY* aND ARTHUR A. Nexson. Div. of Pharma-
cology, Food and Drug Admin., Dept. of Health,
Education and Welfare, Washington, D.C.
Fourteen adult mongrel dogs were fed 100, 50,

25 or 10 mg/kg/day of coumarin; 100 mg/kg/day
were fatal in about 2 wk., whereas 50 mg/kg/day
permitted survival for 1-9 months. At these high
levels and in 1 of 4 dogs on 25 mg/kg/day there
were anorexia, weight loss apathy and weakness,
Gross lesions were jaundice, yellowish livers with
a ‘nutmeg’ appearance and emaciation. The liver
showed a striking microscopic picture of marked
disorganization of the normal lobular pattern,
variation in hepatic cell size (with some irregu-
larly shaped and highly vacuolated cells up to
100 uw in diameter), bile duct proliferation, fatty
change and, to a lesser extent, fibrosis, pigmenta-
tion, erythrocyte inclusions in hepatic cells,
hepatic cell necrosis and intranuclear and cyto-
plasmic inclusions. In 3 more dogs on %
mg/kg/day and in 4 at 10 mg, there were only
slight effects at the 25-mg level and practically
none at 10 mg, after 9-11 months of feeding. Slight,
more or less secondary, changes were present in
various other organs. Rats were less affected by
coumarin than were dogs. Weanling rats fed 2500
ppm in their diet (about 150 mg/kg/day) for 27
wk. showed slight liver changes, whereas those
fed 1000 ppm were negative. In young adult rats
fed 1% in the diet for 4 wk. there was much weight
loss and slight liver injury. Organs other than the
liver were not affected in the rat.

lume 15

ymmani
on tests
rformed
dy rises
ths legs
dy rise,
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th with
y. Sixty
vashings
uses re-
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January
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than the

AMERICAN INSTITUTE OF NUTRITION

Twentieth Annual Meeting
ATLANTIC City, NEw Jersey, Aprit 16-20, 1956

An. asterisk * following an author’s name indicates “by invitation’’

1758. Reversal of sulfaguanidine toxicity in
rat. C. J. ACKERMAN (introduced by W. D.
Satmon). Dept. of Biochemistry and Nutrition,
Virginia Polytechnic Inst., Blacksburg.

Thyroid deficiency symptoms noted in rats fed
sulfaguanidine may be prevented or cured if meat
scraps are included in the diet. Weanling rats fed
a diet containing wheat, casein, alfalfa leaf meal,
Crisco, salts, sucrose, all known vitamins, and
1.1% sulfaguanidine cease to grow after 4-5 wk.
Growth is normal (35-40 gm/wk.) if 0.02% desic-
cated thyroid, or 3% meat scraps are added, or if
sulfaguanidine is omitted. Tentori and Vivaldi
(Rend. ist. super. sanita 17: 19, 1954) reported that
the addition of feces from normally fed rats
prevented the appearance of the syndrome. Using
a curative-type assay, we find that rat, cow, or
chicken feces’ had no beneficial effect. That the
substance in meat scraps may be thyroxine is now
under investigation. The procedure of Kendall
(Thyroxine New York: Chemical Catalog Co.,
1929) for the isolation of thyroxine was applied to
fresh thyroid tissue and meat scraps. All fractions
and the crude thyroxine precipitate obtained from
the thyroid tissue promptly stimulated growth in
rats. Comparable fractions obtained from meat
scraps were inactive. Desiccated thyroid refluxed
for 24 hr. with 6 n HCl or 5% NaOH did not lose
activity. Meat scraps refluxed for 24 hr. with
6 n HCl was still active but refluxing for 24 hr.
with 5% NaOH or 36 hr. with concentrated
NH,.OH resulted in complete loss of activity.

1759. Comparative effect of cottonseed oil
and lard on cholesterol metabolism in rat.
Litta AFTERGOOD,* Rostyn B. ALFIN-SLATER
AND Harry J. Deven, Jr. Dept. of Biochemistry
and Nutrition, School of Medicine, Univ. of
Southern California, Los Angeles.

Plasma and liver cholesterol levels were de-
termined in male and female rats kept for 6, 12,
18 and 24 wk. on 15% cottonseed oil or 15% lard
diet with or without the addition of 1% cho-
lesterol. Animals on a diet containing lard con-
sistently exhibited higher levels of liver cholesterol
as well as plasma cholesterol when compared with

541

animals fed a similar diet where cottonseed oil was
the sole source of fat. These differences became
more pronounced with time. In addition, a sex
difference was observed; in females higher plasma
cholesterol levels and lower liver cholesterol levels
obtained on both diets. It was also found that the
cholesterol concentration was higher in the
carcasses of rats fed the lard diet than of those fed
the cottonseed oil diet. At the same time, no
difference was noted in the absorption of cho-
lesterol, or in the biosynthesis of cholesterol by
liver slices, in the animals fed either fat. Total
liver cholesterol was significantly decreased by the
addition of high amounts of vitamin E to the lard
plus cholesterol diet; this effect was not as striking
in the absence of exogenous cholesterol. The
dietary effect was reflected qualitatively in the
liver lipids as well; namely, the unsaturation level
of fatty acids forming cholesterol esters, and the
amount of phospholipids present, were higher on
the cottonseed oil diet. It is suggested that some
of the causes for the accumulation of cholesterol
in the liver, when lard is the source of fat in the
diet, are the lower lability of the more saturated
fatty acid esters of cholesterol and the inadequate
amount of phospholipids present which are
probably necessary for proper cholesterol trans-
port. (Supported by grants from Best Foods, Inc.,
Bayonne, N. J., and the PHS.)

1760. Acetin fats and vitamin A metabolism.
J.C, ALEXANDER* AND F. H. Mattson. Research
Div., Procter and Gamble Co., Cincinnati, Ohio.
The long chain fatty acids of a conventional

type triglyceride may be replaced with one acetic

acid to produce a monoacetin fat or with two
acetic acids to produce a diacetin fat. These tri-
glycerides have properties of interest to the food
industry. Present experiments describe the ab-
sorption, storage, and utilization of vitamin A by
male rats fed purified diets containing 14% of fat.

The control fat was a mixture of soybean oil and

cottonseed oil which had been hydrogenated to

an iodine value of 80. The experimental fat was a

mixture of mono- and diacetin fat derived from

the control fat. Weanling animals were depleted

542

by feeding vitamin A-free diets. Following de-
pletion, supplements of vitamin A were given
orally. Animals were killed a) after 12 hr. to
measure vitamin A absorption, b) after 48 hr. to
measure vitamin A storage, and c) after 2 and 4
wk. to determine vitamin A utilization. Evalua-
tions were based on data obtained by analyses of
the liver for vitamin A content by the antimony
trichloride method. The acetin fat facilitated the
absorption of vitamin A and promoted liver
storage of vitamin A at least as well as the control
fat. Also, with the acetin fat, a normal rate of
utilization of vitamin A from the liver was found.

1761. Determination of nitrogen balance in-
dexes of dietary proteins in cat. J.B. ALLISON,
S. A. Mituter* anp M. K. Brusu.* Bureau of
Biological Research, Rutgers Univ., New Bruns-
wick, N. J.

A preliminary semi-synthetic diet has been
developed for the determination of nitrogen
balance indexes of dietary proteins in the cat.
More of the B complex was needed in the cat diet
than was found necessary to meet the require-
ments for dogs, rats, mice and hamsters. The diet
supported adequate growth in kittens and mainte-
nance in adults. A linear relationship between
nitrogen intake and nitrogen balance was estab-
lished so that indexes could be calculated. These
indexes were determined to be as follows: egg
white, 1.06; casein, 0.90; beef, 0.90; and wheat
gluten, 0.74. Except for egg white, these values are
higher than those found for maintenance in the
adult dog. The value for wheat gluten is nearer to
that reported for maintenance in the rat than for
any other animal. The cat did not always eat the
low protein or protein-free diets adequately so
that, routinely, the so-called endogenous excretion
of urinary nitrogen could be determined by
feeding sufficient.egg white to maintain nitrogen
equilibrium, the index of egg white being close to
unity. The egg white was almost or entirely
digested. The caloric intake for maintenance in
the adult cat under laboratory conditions was
approximately 110 C/day/kg of body weight. The
egg profem- nitrogen for maintenance of equi-
librium was approximately 0.38 gm/day/kg. A
higher protein intake, however, was believed to
improve the welfare of the adult cat.

1762. Serum cholesterol in subjects on re-
ducing diets high and low in fat. JosEPH T.
ANDERSON, HENRY LONGSTREET TAYLOR AND
ANcEL Keys. Physiological Hygiene, Univ. of
Minnesota, Minneapolis.

Twelve obese college students, 9 male and 3
female, were fed 1200 Cal. reducing diets for 9 wk.
Mean body weight loss was 6.6, 4.2 and 3.0 kg in
successive 3-wk. periods. Diets HF and LF

FEDERATION PROCEEDINGS

Volume 16

containing respectively 58 and 18% of calories as
fat were fed to subgroups of 6 each. At the end of
3 wk. the serum cholesterol concentration had
fallen in every subject, the average decrease being
17% for group 1 on HF and 28% for group 2 on LF,
The diets were reversed, and 3 wk. later the serum
cholesterol concentrations averaged 16% below the
pre-diet control for group 2, now on HF, and 23%
below for group 1, now on LF. Weight losses in the
two groups were almost identical. The difference
in serum cholesterol between the groups was
clearly related to the fat content of the diets. The
average serum cholesterol concentration during
subsistence on LF was 19 mg/100 ml lower than on
HF and this difference is statistically significant.
In a subsequent period of 3 wk. the diets were
continued unchanged, both groups continued to
lose weight, but serum cholesterol concentrations
did not change further. The proportion of the total
cholesterol in the beta lipoprotein averaged 81%
before dieting and did not change significantly
during any subsequent period. It is concluded that
caloric undernutrition and low fat diet cause lower
serum cholesterol independently and _ simul-
taneously in obese college students.

1763. Early histologic changes in experi-
mental atherosclerosis. 8. B. ANDRUs,* G. V.
Mann, L. C. Fruuios* anp F. J. Stare. Dept. of
Nutrition, Harvard School of Public Health,
Boston, Mass.

Atherosclerosis occurs in cebus monkeys fed cho-
lesterol and sulfur amino acid deficient diets and
in rats fed cholesterol, cholate and thiouracil. In
both species earliest visible lesions consist of fine
extracellular lipid within the ground substance
adjacent to the innermost elastic lamellae. As lipid
and ground substance accumulate, medial muscle
cells acquire lipid and become hyperplastic and
reoriented radially. Intimal penetration of muscle
cells is apparent as well as hyperplasia of similar
cells along the inner aspect of the internal elastic
membrane associated with formation of new
elastica. These changes precede foam cell activity
and at no stage is endothelial cell activity promi-
nent. Smooth muscle cells are present in all stages
of intimal plaque formation. In older lesions, loss
of muscle cells of the media was seen with thinning
of the latter. Control cebus monkeys commonly
show non-fatty intimal musculo-elastic lesions,
similar to those described as aging phenomenon
in man. These changes resemble many of the
features of the induced atherosclerosis, and the
latter may be superimposed on the former. Though
the sequence of early events outlined above has
not been verified in other species, muscle cell
activity has been seen in cholesterol induced
atherosclerosis in the rabbit and Rhesus monkey,
and in the lesions of both infant and adult humans.

~~ 6: oy ae a ft afk ee ee ee Se ee

oe; <_

ume 16

ries as
end of
ym had
e being
on LF,
- serum
low the
1d 23%
; in the
ference
98 was
ts. The
during
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ificant.
‘S were
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1e total
od 81%
icantly
ed that
e lower
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2*xperi-
*G.V.
Dept. of
Health,

ed cho-
ets and
acil. In
_ of fine
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March 1956

1764. Multiple level studies for estimation of
the anabolic properties of steroids. AARON
ARNOLD, JEssy S. ScHap* anp A. L. BEYLER.*
Sterling-Winthrop Research Inst., Rensselaer,
Wow.

For some years we have been interested in
quantitative comparisons of substances of bio-
logical interest by means of tests with animals.
Oftentimes such tests may be set up so as to yield
straight line responses at multiple levels of ad-
ministration of the test substances. Such straight
line responses serve several useful functions. One,
they supply a basis for meaningful comparisons;
perhaps more importantly, they serve to protect
the investigator from drawing conclusions which
are contrary to fact. In an application of this
approach we have been evaluating the anabolic
activity of steroids by means of nitrogen balance
studies utilizing adult castrated male rats. They
are brought essentially to weight and nitrogen
equilibrium by gradually reducing the daily
allotment of diet which has the following compo-
sition: dextrin 34.07, cerelose 34.07, yeast 9.2,
hydrogenated vegetable oil 7.4, casein with 3%
methionine 6.0, methyl cellulose 5.0, Jones and
Foster salts 3.7, cod liver oil 0.25, wheat germ oil
0.16, choline chloride 0.15 and Wilson Liver
Extract Fraction O, 0.005. Additional bulk is
provided by adding 10 parts of alpha cel/100 parts
of feed. Animals equilibrated with daily weighed
portions of this feed exhibit straight line nitrogen
retentions as the result of increasing doses of
testosterone propionate given in oil subcu-
taneously over a 5-day period. The amount of the
ester given to groups of 5 rats each has been in the
range of 25-250 ug/rat/day. This has permitted the
evaluation of the anabolic properties of com-
pounds of interest.

1765. Pantothenic acid deficiency and hy-
pophysectomy. JoserH J. BARBORIAK (intro-
duced by G. R. Cowa1tu). Nutrition Lab., Yale
Univ., New Haven, Conn.

Pantothenic acid (PA) deficiency results in re-
tardation and eventual cessation of growth in rats;
somatotrophic hormone (STH) does not induce
further growth (BEaRE et al., Endocrinology 55:
40, 1954). To gain further insight into the relation-
ship between pantothenic acid and growth, a
study with hypophysectomized rats was carried
out. Immediately after hypophysectomy groups of
animals were placed on PA-deficient and on
control diets respectively. Beginning 30 days after
the operation, 4 of the rats in both control and
deficient groups were treated with daily injections
of 3.0 mg STH. On the 112th day of the experiment
(82nd day of hormone administration) all sur-
viving animals were killed. The STH administra-
tion resulted in an immediate growth response in

AMERICAN INSTITUTE OF NUTRITION

543

both control and deficient groups; the weight gains
of the deficient group were about 4 those of the
control animals. Mortality was high in the de-
ficient groups; the average survival time of the
STH-injected animals was 31 days, of the un-
injected animals 55 days. The adrenal cortex of
the STH-injected hypophysectomized deficient
rats was congested and hemorrhagic; the relative
size of cortical zones was about normal. The
adrenal glands of the uninjected rats did not differ
significantly between control and deficient groups:
no adrenal lesions have been observed in either
group. These results are interpreted as meaning
that some mechanism other than a pituitary
mediated ‘stress situation’ is probably responsible
for the adrenal damage in pantothenic acid
deficiency.

1766. Inhibitory effect of androgenic steroids
on 38-ol-steroid dehydrogenase activ-
ity of beef adrenal homogenate. A. L.
BeyYLer,* Sarrtey D. SoBeLL* aND AARON
ARNOLD. Sterling-Winthrop Research Inst.,
Rensselaer, N. Y.

Marked inhibition of 38-ol-steroid dehy-
drogenase of beef adrenal homogenates has been
effected in vitro by a variety of androgenic sub-
stances incubated for 2 hr. in the following system:
25 ml equal parts bovine serum and phosphate
buffer (pH 7.4), 2um A®-androstene-38 , 178-diol (sub-
strate), 6um DPN, lum niacin-amide, and 1 ml of
homogenate containing 25 mg beef adrenal tissue
(source of enzyme). The isolation and determina-
tion of the a,8-unsaturated ketone formed during
the period of incubation were conducted es-
sentially in accordance with the procedures
described by Wiswell and Samuels (J. Biol. Chem.
201: 155, 1953). Methylandrostenediol was the
most potent inhibitory androgen tested; a 40%
inhibition of 36-ol-dehydrogenase activity was
observed with 0.2um, 73% with lum and 100% with
5un of this steroid. Other androgenic steroids were
observed to inhibit the adrenal 38-ol-dehy-
drogenase ; however, no correlation between degree
of androgenicity and enzyme-inhibitory capacity
could be demonstrated. No specific structural
requirements were indicated within the series of
androgenic compounds tested. Non-androgenic
steroids such as progesterone, DCA and estrone
were inactive at 20um per flask, whereas 20um
cortisone acetate inhibited the adrenal enzyme to
the extent of 65%. The 38-ol-dehydrogenase
activity of testicular, but not adrenal, tissue
removed from rats pretreated with methyl-
androstenediol for 2 wk. at 2 mg/100 gm/day was
depressed significantly. These observations sug-
gest the possibility that certain androgenic
substances may interfere in steroidal biosynthetic

544

mechanisms requiring 38-ol-dehydrogenase by a
direct inhibition of this enzyme.

1767. Essential fatty acid deficiency in chick.
J. G. Brert, G. M. Briaes, M. R. S. Fox,*
C. J. Potuarp* anp L. O. Ort1z.* Nail. Insts.
of Health, Bethesda, Md.

Day-old New Hampshire chicks were placed on

a synthetic, fat-free diet (20% purified casein, 8%

gelatin, 0.3% methionine, 6% salts, 65.5% glucose,

and all essential vitamins; J. Agric. Food Chem.

3: 436, 1955). In early experiments, performance

was poor due to inadequate vitamin A. When

crystalline vitamin A acetate was increased 10

times to 30 mg/kg diet, growth was normal for

4-6 wk., at which time weights began to fall

behind control birds receiving the above diet plus

4% corn oil. By 14 wk., fat-deficient chicks were

250 gm (15%) lighter than the controls. At 8 wk.

serum analyses for diene, triene, and tetraene

fatty acids revealed (expressed as percentage of

total fatty acids): group I (controls) 19.6, 0, 25.1,

respectively; group II (no fat) 2.3, 10.1, 2.7;

group III (no fat and no vitamin E) 2.7, 10.4, 2.7.

At 11 wk. the diet of 2 chicks in group II was

supplemented with 8.6 gm methyl linoleate/kg

diet, as the urea complex. Serum analyses at 13

wk. for diene, triene, and tetraene fatty acids

revealed: group I, 15.9, 0, 23.2, respectively;
group II (+ methyl linoleate) 9.8, 7.4, 4.3; group

IIT, 1.3, 11.4, 0.8. The only visible signs of fat

deficiency after 18 wk. other than retarded growth,

were a slight decrease in pigmentation of feathers

and dry, scaly skin. Deaths occurring after 18-26

wk. on the fat-free diet were attributed to air sac

infection. Autopsy revealed immature oviducts
and ovaries in the fat-free birds.

1768. Relationship of protein level to mini-
mum lysine requirement of rat. RIcaRDO
BreEssANi* AND Epwin T. Mertz. Dept. of
Biochemistry, Purdue Univ., Lafayette, Ind.
Weanling rats were fed isocaloric diets in which

corn gluten supplied 54% of the nitrogen, purified

amino acids 32%, and diamm. citrate 14%. Except
for lysine; all diets contained the proportions of
essential amino acids found in whole egg protein.

Minimum lysine requirements for maximum

weight gains were determined from a graph plot

by using total gains for 28 days of 3 males and 3

females at each lysine level. A 4% protein level

gave insufficient growth response. Minimum lysine

requirements at higher levels were (in %) 0.55,

0.67, 0.83, 0.84, 0.87, 0.83 and 0.88 with 8, 12, 16,

20, 24, 32, and 40% total protein (N X 6.25),

respectively. Expressed as percentage of the total

protein, the lysine requirements are 6.9, 5.6, 5.2,

4.2, 3.6, 2.6, and 2.2 resp. The data do not agree

with that of other workers (ALmquist, H. J.,

FEDERATION PROCEEDINGS

Volume 16

Arch. Biochem. 44: 245, 46: 250, 1953) who find in
chickens and pigs that amino acid requirements
increase with all increases in level of dietary
protein, but are a constant when expressed as
percentage of the total dietary protein. Since
these investigators determined amino acid re-
quirements with peptide-bound amino acid from
proteins, and we used mainly free L-lysine (at the
40% protein level, gluten provided only } of the
lysine), our data suggest that animals can use free
lysine more efficiently than peptide-bound lysine,
the minimum requirement (expressed as % of the
diet) remaining constant over a wide range of
protein levels. Published data on the minimum
amino acid requirements of various species must
be reassessed in the light of these findings.

1769. Daily nitrogen retentions of aging
women on self-selected diets. Wiuma D.
BREWER, PEARL Swanson, Lipa BurRRILL,*
JANE LEICHSENRING, HELLEN LINKSWILER,*
May ReyNotps AND MarGaReT MANGEL,.*
Agric. Exper. Stations of Michigan, Iowa, South
Dakota, Minnesota, Nebraska, Wisconsin and
Missouri.

Daily intakes and urinary excretions of nitrogen
were determined for 20 women, 34-78 years of age,
on self-selected diets for periods of 26-30 days.
Average daily fecal nitrogens were determined
for continuous 5 or 7 day periods during the study.
Subjects were studied in states of the North
Central region, i.e., Iowa, Michigan, Minnesota,
Missouri, Nebraska, South Dakota and Wisconsin.
Day-by-day variations in nitrogen retention were
found in all cases; however, only 7 women were
essentially in nitrogen equilibrium. Nine women
had nitrogen balances which were predominantly
positive and 4 had nitrogen balances which were
predominantly negative throughout the period.
Nitrogen equilibrium was predicted for the indi-
vidual women at intakes ranging from 6.4 to 15.0
gm/day. The data indicate that adult women have
the capacity to maintain storage or loss of nitrogen
for relatively long periods of time. Standard errors
of the regression coefficient of nitrogen retention
on nitrogen intake were calculated for the indi-
vidual subjects for cumulative 3-day intervals.
The average standard error of the regression
coefficients was decreased during the initial period
of 10-15 days, then remained relatively constant.

1770. Effect of adrenalectomy on response to
isoleucine deficiency in rats. JEss W. Brom-
LEY AND THomas E. SopERBERG* (introduced
by Leo T. SamueEts). Dept. of Biological Chem-
istry, Univ. of Utah College of Medicine, Salt
Lake City.

Intact and adrenalectomized adult male rats
were force-fed complete and isoleucine-deficient

—= is ofpepme = ~& 42 ee &

olUwS

ch;

dil

ine}
frac
phe
frac

velc
the

me 16

ind in
ments
ietary
ed as
Since
id re-
| from
at the
of the
se free
ysine,
of the
ige of
imum
; must

aging
ra D.
RILL,*
ILER,*
NGEL.*

South
n and

trogen
of age,
| days.
mined
study.
North
1esota,
sonsin.
n were
n were
women
nantly
h were
period.
e indi-
to 15.0
n have
trogen
| errors
ention
e indi-
ervals.
ression
period
stant.

nse to
Brom-
oduced
| Chem-
e, Salt

le rats
eficient

March 1956

diets. Four groups were set up as follows: 1) intact,
complete, 2) intact, deficient, $) adrenalectomized,
complete, 4) adrenalectomized, deficient. Within
60 hr. 37.5% of group 4, the adrenalectomized
deficients, were dead without apparent cause. To
determine whether abnormal amino acid metabo-
lism was involved the remaining animals were
anesthetized with sodium Nembutal and ex-
sanguinated. The plasmas of the 4 groups were
then analyzed for Na and K by flame photometer
and for individual amino acids by DNP technique.
The levels of valine, threonine, phenylalanine,
leucine, and methionine in group 4 were much
higher than in any of the other groups. On the
other hand, the levels of glycine, proline, serine,
and tryptophane were the same. The totals of the
13 amino acids in group 4 were more than double
the totals of the same 13 amino acids in any one
of the other three groups. There was, therefore, a
specific effect on accumulated amino acids. Data
regarding the amino acid levels will be presented,
and the significance of the changes occurring in
the above 4 groups will be discussed.

171. Distribution of a labeled lipid in ali-
mentary lipemia. WILLIAM W. Burr, JR.* AND
Hersert C. Tipwewu. Biochemistry Dept.,
Univ. of Texas, Southwestern Med. School, Dallas.
In earlier studies palmitic acid-1-C™ dissolved

in olive oil was fed by tube to white rats and the

changes in blood particulate fat and radio-

activity followed. As an extension of this work a

study has been made of the relationship of the

radioactivities of the chylomicron and nearly
chylomicron-free blood fractions at various
intervals following administration of the radio-
active fat. The C'4-labeled fat was given orally
and blood was obtained periodically from the tail
vessels for determination of radioactivity and
chylomicron counting. After 13, 4 and 5 hr., the
animals were killed by collecting blood by cardiac

puncture and the blood was diluted with a 20%

urea solution. Chylomicron counts and assays for

radioactivity were made on a sample of the
diluted blood and on an aliquot after it was

subjected to centrifugation for 90 min. at a

minimum of 26,500 X g. An increase in the radio-

activity of the infranatant fraction appeared to
occur with an increased duration of the experi-
ment. The possibility that this represents an
increasing amount of activity in the lipoprotein
fraction has been investigated with paper electro-
phoretic techniques and lipid recoveries from this
fraction. (Supported in part by a grant from the

Lipotropic Research Fndn. and contract DA-49-

007-MD-662 with the Div. of Research and De-

velopment, Office of the Surgeon General, Dept. of

the Army.)

AMERICAN INSTITUTE OF NUTRITION

545

1772. Digestibility of modified fats. Doris
Howes CatLoway AND GEorGE W. Kurtz.*
GM Food and Container Inst., Chicago, Ill.

In order to investigate the relationships among
melting point, saturation, chain length, structure
and digestibility a series of natural and modified
fats have been fed to mature rats. Fat (20%) was
incorporated into a purified diet which was fed for
2 wk. Feces were collected during the last 5 days
for fat analysis. The natural fats studied, repre-
senting wide variation in saturation and chain
length, were: cottonseed, soybean, corn, coconut.
and palm oils, lard and butterfat. Digestibility of
these fats was uniformly high and unrelated to
the characteristics cited. When these same fats
were fully hydrogenated, digestibility varied
inversely in linear fashion with melting point and
with chain length of the constituent fatty acids,
and in curvilinear manner with stearic acid
content. Although similar in melting point, the
monoglyceride of hydrogenated lard was more
digestible than was the triglyceride. Equally
effective in enhancing digestibility was substitu-
tion of 4 of the fatty acid radicals by butyryl
groups; however, in simple mixture tributyrin did
not benefit the absorption of triglyceride lard.
These findings suggest that digestibility is de-
pendent upon the chain length of the saturated
fatty acids and their arrangement within the
glyceride structure.

1773. Estimation of metabolizable energy of
cereal products from their chemical com-
position. K. J. CARPENTER AND K. M. CLEeae
(introduced by D. M. Hrasrep). Rowett Research
Inst., Abderdeen, Scotland.

Estimates of the metabolizable energy (m.e.)
of feedstuffs or mixes may be needed when analysis
can be undertaken but not a feeding-trial. To test
possible formulae for predicting m.e. for poultry,
samples of cereals, cereal by-products and mixed
rations were fed (with suitable proteins, minerals
and vitamins) to laying hens and their m.e. de-
termined from calorimetry of feed and excreta.
Values for 17 samples (corrected to 90% dry
matter), covered a range from 1300 to 3500 Cal/kg.
In trying to estimate these values from the results
of conventional proximate analysis, the best
relation found was expressed by ‘m.e. =
120 (% crude protein + 2.25 X % ether extract +
nitrogen-free extractives) — 7,390’ with a S.D. of
+340 Cal/kg. On replacing the ‘residual’ nitrogen-
free extractives term with direct starch and sugar
values obtained with a simple anthrone method
modified for use with feedstuffs (CLEGG, Biochem.
J. 61: xvii, 1955) a closer relationship was found,
expressible as ‘m.e. = 38 (% crude protein +
2.25 X % ether extract + 1.1 X % starch + %
sugar) + 53’ with aS8.D. of +190 Cal/kg. Some of

546

the deviation can be attributed to errors in the
direct determination of m.e.; the remainder may
come from variable digestibility of protein and
fat and a small contribution from digestible
carbohydrates other than starch and sugar (see
Botton, J. Agric. Sci. 46: 119, 1955). The extreme
grades of wheat offals tested had 2,650 and 1,320
Cal/kg. of m.e. They contained 61 and 55%
respectively of nitrogen-free extractives, but the
former had 43% of ‘starch + sugars’ and the latter
only 18%. (Supported in part by the Kellogg
Fndn.)

1774. Relative roles of niacin and tryptophan
in maintaining pyridine nucleotides, nitro-
gen balance and_= growth. Marilyn
CuHaLoupKa,* J. N. WILLIAMs, JR.* AND May S.
ReyNo.tps. Dept. of Biochemistry and School of
Home Economics, Univ. of Wisconsin, Madison.
Young adult rats, depleted of blood pyridine

nucleotides (PN) by a niacin-tryptophan deficient

ration, were fed supplements of L-tryptophan
from 0 to 0.30% of the ration. Nitrogen equilibrium
was achieved on the lowest levels of tryptophan
supplementation, growth on the intermediate
levels, and synthesis of blood PN on the higher
levels. This sequence suggests a preferential use
of tryptophan in the order listed by niacin-
tryptophan depleted rats. When similarly depleted
rats were fed constant physiological levels of
tryptophan, niacin, or tryptophan plus niacin,
those receiving niacin showed a further decrease
in blood PN, those receiving tryptophan main-
tained a constant level of blood PN, and those
receiving both nutrients showed a definite in-
crease in blood PN. This supports the suggestion
that at physiological levels tryptophan contrib-
utes to PN synthesis to a greater extent than
niacin, although niacin in combination with
tryptophan, produced the greatest PN synthesis.

1775. Metabolism of radioactive nicotinamide
in chick. M. L. Wu Cuana* anp B. Connor
JOHNSON. Dept. of Animal Science, Univ. of
Illinois, Urbana.

Following the injection of C!*-carboxyl-labeled
nicotinamide into chicks which had been operated
on so as to be able to separate urine from feces,
the radioactive metabolites in the urine were
separated by paper chromatography and detected
by radioautographs. 6-Nicotinyl-p-glucuronic
acid, nicotinuric acid, 5-mononicotinylornithine,
a-mononicotinylornithine, nicotinic acid, 2,5-
dinicotinylornithine and nicotinamide were iden-
tified in the urine. Liver, kidney, spleen, and
light and dark muscles have been similarly
studied. The radioactive metabolites found in
urine were also found in the liver and kidney
but only nicotinamide was found in the muscles.
The methods of identification will be discussed.

FEDERATION PROCEEDINGS

Volume 1§

1776. Effect of x-irradiation on distribution
of fatty acids in rat tissues. AMBER L. §,
CHENG,* SupRAVAT MUKHERJEE,* Rostyn B,
ALFIN-SLATER AND Harry J. DE5EUEL, JR,
Dept. of Biochemistry and Nutrition, School of
Medicine, Univ. of Southern California, Los
Angeles.

The effect of x-irradiation on the fatty acid
distribution in the tissues of rats on two experi-
mental dietary regimens has been studied. Wean-
ling male rats were evenly divided into two
groups and placed on a 0% fat diet and a 15%
cottonseed oil diet for 13-16 wk. at which time
the weight of the animals on the 0% fat diet had
reached a plateau. At this time, the animals
were further divided and half of each group was
subjected to a whole body x-irradiation of 6001,
The animals were killed 7 days after exposure to
x-ray and the skin, heart, liver, intestine, blood
and adipose tissue were excised and extracted to
remove lipids. The extracted lipids were sepa-
rated on a silica-gel column into three compo-
nents—cholesterol esters, triglycerides, and
phospholipids. Animals on both diets subjected
to x-irradiation exhibited marked changes in
the fatty acid pattern of skin, adipose tissue and
liver; these effects were significantly greater in
the fatty acids of the animals on the fat-free
diet. In the blood of animals on the 15% cotton-
seed oil diet exposed to x-irradiation, there was
no change in the fatty acid distribution in the
triglyceride and cholesterol ester fractions, but
change in the fatty acids of the phospholipid
fraction occurred—with a general increase in the
more saturated fatty acids at the expense of the
more unsaturated fatty acids. This picture ob-
tained in the lipid fractions of all the tissues
which were affected by the exposure to x-ray. A
possible explanation will be discussed. (Sup-
ported by a grant from the Atomic Energy Com-
mission.)

1777. Lysine requirement of adult men and
women. HELEN CLARK,* Eva Kwona,* JEAN
Howe,* Donatp C. DELoNG* anv Epwin T.
Mertz. Depts. of Foods and Nutrition and Bio-
chemistry, Purdue Univ., Lafayette, Ind.

The quantities of lysine required for nitrogen
equilibrium by healthy men and women between
the ages of 20 and 30 yr. and weighing 50-86 kg
have been investigated. The basal diet contained
159 gm of white wheat flour and 21 gm of cor
meal per day (in the form of baking powder
biscuits and corn bread). This was supplemented
with essential amino acids so that the total quan-
tities approximated those in 20 gm of egg protein.
More than } the total intake of each essential
amino acid was derived from protein. The basal
diet contributed 450 mg of lysine. Each subject
consumed 9.0 gm of nitrogen daily, of which 4.4]

col

of
sea

177

lume 1§

bution
R L. §.
LYN B,
1L, Js
chool of
ta, Los

ty acid
experi-
. Wean-
ito two
| a 15%
ch time
liet had
animals
up was
of 600 r,
osure to
e, blood
acted to
re. sepa-
compo-
s, and
ibjected
nges in
ssue and
eater in
fat-free
- cotton-
ere Was
1 in the
is, but a
pholipid
e in the
e of the
bure ob-
. tissues
x-ray. A
|. (Sup-
zy Com-

en and
},* JEAN
DWIN T.
and Bio-

nitrogen
between
50-86 kg
ontained
of corn
powder
emented
al quan-
protein.
essential
he basal
. subject
vhich 4.0

March 1956

gm were supplied from foods, 0.8 gm from an
amino acid mixture, and 4.2 gm supplied equally
from glycine, glutamic acid and diammonium
citrate. The daily caloric intakes of the women
(30-46/kg) and of the men (44-51/kg) were suffi-
cient for maintenance of body weight. Retention
of nitrogen in 7 subjects followed intakes of lysine
between 500 and 700 mg. An 8th subject was in
equilibrium with 600 mg in one experiment, but
was not in equilibrium with 700 mg in a subse-
quent test after ingestion for 20 days of a diet
containing suboptimal quantities of lysine.
Another subject treated similarly for 20 days was
in negative balance with 900 mg of lysine. With
the exception of these two individuals, the lysine
requirements observed to date with a diet con-
taining substantial amounts of protein fall within
the range reported by Rose (J. Biol. Chem. 214:
579, 1955) using mixtures of purified amino acids.
(Contribution of North Central Regional Project
NC-5.)

1778. Transplantation of cholangiofibrosis
lesion from liver of chronic choline-de-
ficient rat. D. H. CopeLtanp,* W. D. SALMON
AND M. J. Burns.* Dept. of Animal Husbandry
and Nutrition, Agric. Exper. Station, Alabama
Polytechnic Inst., Auburn.

Certain choline-deficient diets produce exten-
sive cholangiofibrosis in the livers of rats. Be-
cause of the size and the atypical microscopic ap-
pearance of some of these lesions, transplantation
has been attempted. The liver of chronic choline-
deficient female AES rat No. 31067 exhibited
several large, grossly-visible areas of cholangio-
fibrosis. (Later microscopic examination of these
lesions revealed only cirrhosis and atypical
cholangiofibrosis.) One area was excised and
transplanted subcutaneously into six 6-day-old
AES rats. A tumor was found at the transplanta-
tion site in the individual rats at intervals of 4,
6, 6, 7, 8 and 9 months respectively. This tumor
has been successfully transplanted through 6
transplant generations and is now growing in the
7th. The incidence of takes by generations has
been 6/6, 5/6, 7/7, 6/6, 7/7, 8/8, 9/10. The rate
of growth has increased so that since the 3rd
generation the transplants reach an average size
of about 3 cm in 3-4 weeks. The tumors in all
transplant generations appear to be _ fibrosar-
comas and do not show any epithelial ducts or
cords, such as were seen in the original lesion.
The histology of the lesions will be illustrated
and discussed. (Supported in part by grants from
American Cancer Society upon recommendation
of the Committee on Growth of the Natl. Re-
search Council and from the Natl. Cancer Inst.)

1779. Effect of growth hormone and vitamin
D on rats on a low-Ca normal-P diet.

AMERICAN INSTITUTE OF NUTRITION

547

Joun W. Cramer,* Esa I. Porrata-Dorta*
AND Harry STeensock. Dept. of Biochemistry,
Univ. of Wisconsin, Madison.

A study has been made of the comparative
effects of growth hormone and vitamin D on
growth and calcification in rats on a vitamin
D-free, semisynthetic, low-Ca (0.02%), normal-P
(0.8%) diet. Groups of young male albino rats
weighing 80 gm were used. One group was given
only the basal diet; a second group was given the
basal diet plus vitamin D; and a 3rd group was
given the basal diet plus growth hormone and no
vitamin D. Ca and P balances were obtained
from the 4th through the 15th day of the experi-
ment. Bone ash, serum Ca, serum inorganic P,
and metaphyseal widths were obtained with the
termination of the experiment on the 15th day.
It was found that both growth hormone and
vitamin D produced increases in body weight,
femur lengths, femur organic content, and meta-
physeal widths. The increase in body weight
with growth hormone, however, was only 40% of
that produced by vitamin D. While P balances
generally reflected the relative increases in
growth, the Ca losses, which were decreased by
vitamin D, were not affected by the growth hor-
mone. Only vitamin D increased the absorption
of the small quantities of Ca in the diet. And only
vitamin D increased serum Ca and serum inor-
ganic P. While both vitamin D and growth hor-
mone had an effect on growth and therefore on N
and P metabolism, a growth hormone effect on Ca
metabolism was not demonstrated with our
technique.

1780. Heterologous transplantion of leukemic
cells. M. K. Daaa* anv JoserH H. BuRCHENAL.
Div. of Exptl. Chemotherapy, Sloan-Kettering
Inst. and Cornell Univ. Med. College, New York
City.

Transplantable mouse and rat leukemias have
been grown for several generations in the cheek
pouches of cortisonized hamsters. Leukemia
L1210 (dba mice) obtained from Law, has been
carried for 16 generations in cortisone-treated
hamsters. Metastases to liver and spleen have
been demonstrated histologically and by bioas-
say. Slight growth was noted in first generation
transplants into untreated hayasters. This was
improved after passage for 10 generations through
cortisonized hamsters. At the 8th generation the
tumor was transplanted into untreated dba
mice, C58 X Bagg albino Fl mice, AKR mice,
and embryonated eggs. There were no growths in
the eggs. Tumors grew in all mice but regressed
in the Fl and AKR mice. Leukemia 8174 (C58
mice) has been carried through cortisone-treated
hamsters for 9 generations. Optimal growth
period for leukemia 8174 is approximately 20
days in the hamster in contrast to 10 days for

548

leukemia L1210. Leukemia 82 (C58 mice) grew
for 3-5 generations in cortisonized hamsters, but
in 3 separate attempts, this strain could not be
carried in cortisonized hamsters beyond 3-5
generations. The rat monocytic leukemia ob-
tained from Heinle has been serially transplanted
in the cortisonized hamster for 4 generations.
Over 60 sternal marrow aspirations from pa-
tients with acute leukemias both in cortisone
sensitive and resistant stages have shown no
evidence of growth in the cheek pouch of the
cortisonized hamster. Further studies on hetero-
transplantability of human leukemic cells from
marrow and from strains isolated in tissue cul-
ture by Osgood, will be reported.

1781. Anemia in vitamin E-deficient monkeys.
Paut L. Day anp James S. Dinnina. Dept. of
Bochemistry, Univ. of Arkansas School of Med-
icine, Little Rock.

Eight young rhesus monkeys (Macaca mulatta)
were given a purified diet deficient in vitamin E.
Four of these received the diet deficient also in
pyridoxine. All of the animals developed a syn-
drome characterized by progressive muscular
weakness, creatinuria, increased output of uri-
nary allantoin, anemia and mild leucocytosis.
These evidences of tocopherol deficiency appeared
in 5-10 months and the time of appearance was
unrelated to the absence or presence of pyridox-
ine in the diet. A typical anemic monkey showed
the following blood picture on the 166th day:
RBC 2.9 million/ul, hematocrit 30%, hemoglobin
6.4 gm, WBC 27 thousand/ul, neutrophils 96%.
The monkey was given 20 mg of a-tocopherol
phosphate by subcutaneous injection. There was
a 22.8% reticulocyte response 4 days later. On
the 19th day after treatment the blood picture
showed: RBC 4.54 million/ul, hematocrit 44%,
hemoglobin 10 gm, WBC 16.6 thousand/yl and
neutrophils 65%. The following blood data were
obtained on another vitamin E-deficient monkey
on the 225th day of experiment: RBC 1.56 mil-
lion/ul, hematocrit 15%, hemoglobin 4.0 gm,
WEC 11,9 thousand/ul, neutrophils 51%. Thirty-
two days prior to this the WBC had been 35.1
thousand/yzl and the neutrophils 90%. This ani-
mal was killed for tissue study on the 225th day.
The anemia in these animals appears to be a
specific manifestation of tocopherol deficiency.

1782. Classification of essential amino acids
for chinook salmon. Donatp C. DELOoNG,*
Joun E. Hatver* ano Epwin T. MErTz.
Salmon Nutrition Lab., U.S. Fish and Wildlife
Service, Cook, Wash. and Dept. of Biochemistry,
Purdue Univ., Lafayette, Ind.

No studies have as yet been reported on the
classification of essential amino acids for fish. In

FEDERATION PROCEEDINGS

Volume 16

the present experiment, hatchery Chinook salmon
(Oncorhynchus tschawytscha) were divided into
lots of 200 each, one lot per trough, average
weight of individuals per lot, 1.9-2.0 gm. Two
lots were fed a control diet developed previously
(J. E. Hatver, Federation Proc., this issue) con-
taining (in %), L-amino acid mix 35, carboxy.
methylceliulose 5, dextrin 3, corn oil 2.5, minerals
2, vitamin-cellulose 1.5, cod liver oil 1, and water
50. Other lots were fed a modification of the con-
trol diet in which a-cellulose flour replaced one of
the 18 purified amino acids. At the end of 6 wk.
the average total weight increases of individuals
per lot were (in gm), control 0.76, control 0.83
(lots 3-20, resp. devoid of): arg. —0.1, his. 0.1,
ileu. 0.0, leu. —0.2, lys. 0.0, met. 0.1, phe. 0.1,
thr. 0.0, try. 0.0, val. 0.0, ala. 0.75, asp. 0.85, cys,
0.83, glu. 0.94, gly. 0.95, pro. 0.85, ser. 0.89, tyr.
0.78. At the end of 6 wk. lots 3-12 inclusive were
divided into 2 equal sub-lots, one to be continued
as before, the other placed on the control diet. A
prompt growth response was observed in all sub-
lots returned to the control diet. Data at the end
of 10 wk. on all lots and sub-lots paralleled and
confirmed the 6-wk. data. It is concluded that
the Chinook salmon requires the same 10 amino
acids required by the weanling rat and pig,
namely, arginine, histidine, isoleucine, leucine,
lysine, methionine, phenylalanine, threonine,
tryptophan and valine. The salmonid differs from
the rat and pig, however, in its failure to syn-
thesize any significant amounts of arginine.

1783. Comparative effects of DPPD and other
antioxidants on hypervitaminosis A in the
rat. H. J. DEVEL, Jr., R. P. Cox,* R. B. At-
FIN-SLATER AND B. H. Ersnorr. Dept. of Bio-
chemistry and Nutrition, Univ. of Southern
California, Los Angeles.

Weanling male and female rats were fed a puri-
fied basal diet containing all known nutrients in
adequate amounts. The experimental groups
received the basal diet plus synthetic vitamin A
acetate at levels of 1, 3 or 6 million U.S.P. units/
kg of diet with and without DPPD (N,N’-di-
phenyl-p-phenylenediamine), Santoquin or alpha
tocopherol acetate. The relative effects of these
supplements were studied with respect to growth,
occurrence of spontaneous fractures, changes in
the lipid chemistry of the blood, liver and kidneys
and the vitamin A content of these tissues. DPPD
when added at a level of 0.025% or 1% of the diet
accentuated symptoms of hypervitaminosis A as
manifested by the earlier and more marked re-
tardation in growth, the earlier occurrence and
greater incidence of leg fractures, and increased
vitamin A levels in the blood, liver and kidneys.
In contrast to the effects obtained with DPPD,
alpha tocopherol acetate at levels of 0.05% or

@a fn of &

nit
th
tio
the

olume 16

< salmon
1 into 2%
average
m. Two
eviously
ue) con-
carboxy-
minerals
id water
the con-
d one of
of 6 wk.
lividuals
trol 0.83
his. 0.1,
he. 0.1,
).85, cys,
.89, tyr.
ive were
ontinued
| diet. A
all sub-
the end
sled and
led that
0 amino
nd pig,
leucine,
reonine,
ers from
to syn-
ne.

d other
. in the
, B. At
of Bio-
Southern

| a puri-
‘ients in
groups
‘amin A
. units/
1, N’-di-
yr alpha
of these
growth,
nges in
kidneys
. DPPD
the diet
jis A as
‘ked re-
nee and
creased
cidneys.
DPPD,
05% or

March 1956

0.5% of the diet or Santoquin at a level of 0.05%
of the diet resulted in little if any increase in the
symptoms of hypervitaminosis A when fed in
conjunction with massive doses of this vitamin.

1184. Metabolic disposal of folic acid. JAMES
§. Dinnine, Joun T. Srme* anp Paut L. Day.
Dept. of Biochemistry, Univ. of Arkansas School
of Medicine, Little Rock.

Incubation of folic acid (pteroylglutamic acid,
PGA) with rat liver slices results in the enzy-
matic formation of a diazotizable amine. This
amino formation is completely inhibited by 4-
amino-pteroylglutamic acid (aminopterin). When
PGA is incubated with liver homogenates there
is no amine formation. Incubation of synthetic
citrovorum factor with liver homogenates re-
sults in the enzymatic formation of a diazotizable
amine and this reaction is not inhibited by amino-
pterin. The excretion of diazotizable amine in
the urine by rats is proportional to the dose of
injected PGA. The results suggest that PGA is
converted to citrovorum factor which in turn is
cleaved to para-aminobenzoylglutamic acid and a
pteridine moiety. The magnitude of the reaction
suggests that this represents a major pathway
for the metabolic disposal of PGA.

11785. Relationships of vitamin B,:, methi-
onine and choline to metabolism of B
vitamins in hepatic tissues. CecILE HOOVER
EDWARDS AND CHARLOTTE E. OUTLAND (in-
troduced by Peart P. Swanson). Carver Fndn.
and School of Home Economics, Tuskegee Inst.,
Ala.

The concentrations of vitamins of the B group
in hepatic tissue of adult rats have been deter-
mined after the animals received by stomach tube
rations deficient either in protein, certain amino
acids, or vitamin Bie. The effects of supplements
of 8 essential amino acids (not including me-
thionine and arginine), vitamin Bis, methionine
and choline, fed singly and in combination the
latter 9 days of the 24-day experiment, were in-
terpreted on the basis of vitamin:nitrogen ratios
in hepatic tissues. In the absence of dietary pro-
tein, hepatic biotin concentrations and biotin:
nitrogen ratios were normal when supplements
of choline and vitamin Bi2 were provided. Under
these conditions, however, folic acid concentra-
tions and ratios to total nitrogen were higher than
those of normal animals. Vitamin Bi: appeared
to stimulate the transfer of pantothenic acid
from hepatic tissues when the ration was de-
ficient in methionine. Methionine also produced
this effect when dietary protein and vitamin Bie
were restricted. When dietary labile methyl
groups were inadequate, supplementary vitamin
By. stimulated the deposition of excess p-amino-

AMERICAN INSTITUTE OF NUTRITION

549

benzoic acid. Methionine deficiency resulted in
lower hepatic pyridoxine:nitrogen ratios. No
effect on vitamin By2:nitrogen ratios was ob-
served when either protein, methionine, choline
or vitamin Bi. was omitted from the rations.
(Supported in part by a grant from the Nutrition
Fndn.)

1786. Nutritional studies with rats subjected
to thyrotoxic stress. GLapys A. EMERSON,
BarBarRa Esser* anp Atwoop C. Pags.*
Merck Inst. for Therapeutic Research and Re-
search Labs., Chemical Div., Merck & Co., Rah-
way, N. J.

Ershoff reported the need for an unknown fac-
tor(s) by rats fed a purified diet supplied with
the known nutrients but containing thyroid sub-
stance (Arch. Biochem. 15: 365, 1947; Proc. Soc.
Exper. Biol. & Med. 74: 391, 1950). Several ma-
terials, including whole liver insolubles, were
found to be potent sources of this unidentified
factor(s). The original ration of Ershoff has
been modified in this laboratory by changes in
the basic constituents and by replacing 0.5%
thyroid powder with 0.1% protamone (iodinated
casein). The test period has been shortened from
90 to 13 days. The growth of weanling male rats
receiving this modified diet| supplemented with
10% liver insolubles was ca. 30-40% above that
of the unsupplemented controls. Three-quarters
of the response observed when liver was fed
could be obtained by a combination of the fol-
lowing: /) increasing the vitamin-free casein of
the diet from 24 to 34%; 2) replacing 5% of the
10% hydrogenated fat by corn oil and possibly
by increasing the water-soluble vitamins (which
had been supplied in the basal ration at about
10 times the level required by the normal rat);
8) the addition of 0.17% bile acids or cholesterol
to the diet. The remainder of the response was
obtainable by supplementation with liver or
with isolated soybean protein.

1787. Multiple congenital abnormalities re-
sulting from pantothenic acid deficiency in
the rat. Herspert M. Evans,* Marsorre M.
NELSON, CATHERINE D. C. Batrp* aNnD HowarD
V. Wricut.* Inst. of Exptl. Biology, Univ. of
California, Berkeley.

Stock female rats of the Long-Evans strain,
60-65 days of age, were placed on a purified
pantothenic acid-deficient diet the day of breed-
ing or were bred after 4-20 days of deficiency.
When the deficiency was instituted at the begin-
ning of the gestation period, fetal death with
resorption of the entire litter occurred in 22%
of the pregnancies. Macroscopic abnormalities
were observed in 14% of the living young. When
the deficiency was instituted 4-10 days prior to

550 FEDERATION
breeding, the incidence of abnormal young in-
creased to 23% and the incidence of resorptions
to 63%. All animals resorbed with 20 days of
deficiency prior to breeding. The addition of
pantothenic acid to the deficient diet during the
gestation period reversed all effects of the previ-
ous 20 days of deficiency and fetal development
was normal. The abnormalities observed included
those previously reported by other investigators;
namely, encephalocele, hydrocephalus, microph-
thalmia, anophthalmia, edema and digital hemor-
rhages. In addition, defects of the interventricu-
lar septum of the heart and of the aortic arch
pattern, hydronephrosis and hydroureter, ab-
normal position of the gonads, cleft palate, club-
foot and diaphragmatic defects were observed.
Studies with a transitory pantothenic acid de-
ficiency, induced by the addition of x-methy]l
pantothenic acid to the deficient diet during the
gestation period, resulted in the same types of
anomalies.

1788. Dietary protein level and regulation of
food intake. Pau. F. Fenton. Brown Univ.,
Providence, R. I.

The suggestion has been made that caloric
intake is regulated in part by the SDA of the
ration. To test this hypothesis, young adult
male mice of several strains known to differ in
their voluntary food intake were fed a 15% pro-
tein diet. Food intake was measured after the
animals had become accustomed to this ration.
The animals were then fed successively 10, 5 and
3.5% protein diets. At each protein level, volun-
tary food intake was measured after an initial
adjustment period. Daily food intake of all four
strains studied increased as the dietary protein
level was lowered to 5%. Further reduction in
protein level to 3.5% caused no further increase
in food consumption of C3H and A strain mice.
The I and C57 strain mice, however, consumed
less of the 3.5% than of the 5% protein diet. In
another experiment young adult males of these
four strains were accustomed to a 30% protein
diet and voluntary food intake measured. The
dietary,protein level was then increased success-
ively to 50, 70 and 90%. Food consumption was
not greatly affected by these changes in dietary
protein level. Changes in body weight, carcass
composition, and nitrogen excretion have also
been measured. Some effects of dietary protein
level on glucose tolerance have been observed.

1789. The turkey embryo eye as related to a
deficiency of vitamin E. T. M. Ferauson,*
R. H. Riapon* anp J. R. Coucu. Depts. of
Poultry Husbandry and Biochemistry, Texas A.
and M. College, College Station, and Dept. of
Pathology, Univ. of Texas Med. School, Galveston.

PROCEEDINGS Volume if

Mar

Results from this laboratory (Proc. Soc. Exper,
Biol. & Med. 86: 868, 1954; J. Nutrition 55: 387/liffe
1955) and others (Proc. Cornell Nutr. Conf. p. 62
1953; Poultry Sci. 34: 1203, 1233, 1955) have sho
that vitamin E is an important factor for turke
embryonic development. These results were bas
on the addition of vitamin E to a practical di
or a torula yeast diet. The present report con-
cerns the effect of vitamin E supplementation t9
a synthetic-type diet upon embryonic develop.
ment of Beltsville Small White turkeys. Grow
one (Beltsville Small White Turkey hens) was
fed the basal diet and group two, the basal diet
supplemented with D-a-tocopheryl acetate (2)
mg/lb). Live embryos were removed from both
groups of eggs for gross and histological study
from the 5th day of incubation through to hateh-
ing. The tocopherol content of egg yolks after 5
wk. on experiment averaged 130 y/yolk for the
basal group and 797 y/yolk for the E-supple.
mented group. (Analyses courtesy Distillation
Products Industries, Rochester, N. Y.) Opacities
in the eyes occurred in 38.8% of the embryos fed
the basal diet and keratoconus was frequent,
Microscopic studies of the lens from E-deficient
embryos showed various degrees of liquefaction
of the lens fibers, and degeneration of cells of the
lens epithelium. Only 3.6% of the eyes from the
E-supplemented group exhibited opacities in
the lens, and no keratoconus was observed in
this group.

ile
sion

1790. Experimental atherosclerosis in the rat.
L. C. Friuros*, 8. B. ANpRus,* G. V. MANN AND
F. J. Stare. Dept. of Nutrition, Harvard Schol
of Public Health, Boston, Mass.

Purified diets supplemented with cholesterol,
sodium cholate and thiouracil were fed to adult
albino rats for periods up to 363 days. In a few
weeks a marked hypercholesteremia, hyper-
betalipoproteinemia, hepatic lipodiosis and cho-
lesterosis was observed. Grossly sudanophilic§ ,.
lesions were found in all 46 animals examined.
These were most prominent in the heart valves
and aortic arch. The earliest lesions which were
seen at 31 days required Sudan staining for gross
demonstration. Older lesions were visible without
staining. Microscopic coronary artery lesions
were present and in one instance were accom-f,
panied by massive myocardial infarction. Vascu-
lar lesions were characterized by medial and in-
timal lipid infiltration and cellular intimal plaque
formation. In a part of this study the protein
level of the above diet was altered at the expense
of sucrose. The hypercholesteremic response
among 32 rats, divided into 4 dietary groups,
varied according to the protein level. The lowest
response was observed among those animals re-
ceiving the highest level of dietary protein. A

Volumeil
_— March 1966

oc. Exper, : ;
n BB: 3g7 lifference, ‘however, in the severity and extent
of the arterial lesions among these relatively
all groups of rats could not be established. In
| these experiments a close correlation existed
tween the serum cholesterol levels and -lipo-
roteins of the S; 20-100 range. Neither age, sex
wor body weight appeared to influence the lipid
wt vascular responses under these experimental
fonditions.

91. Growth responses of spaced-fed rats.
J. S. Frntayson* anp C. A. Baumann. Dept.
of Biochemistry, Univ. of Wisconsin, Madison.
Weanling rats were trained to eat for only 2
ir/day, and their responses to various nitroge-
ous substances determined. Spaced feeding in-
weased the toxicity of several compounds: die-
lary urea depressed growth in both spaced and
wthodox experiments, but 5% of urea fed 2 hr/
lay was as effective as 30% fed ad libitum. The
epression in growth seemed to depend upon the
nte of urea intake and the level of urea tempo-
nrily attained in the blood; it was not affected
ty the level or adequacy of the protein in the
jasal diet. Spaced feeding increased the toxicity
ff ammonium carbonate, diammonium citrate,
leucine and 2,4-dinitrotoluene. Growth depres-
ions by the ammonium salts varied directly
vith blood urea. Spaced feeding seemed to lessen
the toxicity of 3’-methyl-4-dimethylaminoazo-
benzene and ethanol, but had little effect on rela-
ive growth rates when biotin, vitamin Bis, and
folic acid were omitted from the diet, when gly-
tne was added, or when antibiotics were added
it various combinations to diets limiting in pro-
tin. The procedure was employed successfully
9 determine the biological values of proteins,
amd it may be of value in measuring biological
values when only small amounts of test material
we available.

cal study
to hateb-
cs after}
k for the
K-supple-
istillation
Opacities
bryos fed
frequent,
-deficient
uefaction
lls of the
from the
cities in
erved in

the rat.
[ANN AND
ard Schol

lesterol,
to adult
In a few

hyper-
and cho-
inophilie
camined,
t valves
ich were
for gross
without

lesions
accom-
. Vaseu-
and in-
] plaque
protein
expense
‘esponse
groups,
2 lowest
nals re-
tein. A

1192. Level of protein in diet and its biological
value. R. M. Forpes, MartHa YOHE* AND
LuctteE VauGHAN.* Animal Nutrition Div.,
Univ. of Illinois, Urbana.

The biological value of 3 proteins was deter-
nined by the Thomas-Mitchell method, employ-
ing 6 levels of whole extracted egg protein (4-
4%), 10 levels of casein (4-28%), and 7 levels of
fanut protein (4-25%). Protein was added to
the diets at the expense of carbohydrate, and all
diets contained 10% ether extract, 4% minerals,
md 2% fiber. Five or six rats were used on each
kvel of protein, except that 10 rats were used
mn each of the three lowest levels of egg. The
me digestibility of the proteins was not affected
by protein concentration in the diet. Except for
‘gg protein at 4 and 9% of the diet, the biological
value decreased linearly as the protein concen-

AMERICAN INSTITUTE OF NUTRITION 551

tration increased above 4%. The regression equa-
tions (Y = biological value, X = % protein in
diet) for the linear portion of the egg protein
data and for all the casein and peanut protein
data are, Y = 126 — 2.73X, Y = 96 — 1.78X and
Y = 75 — 1.85X. Statistical analysis reveals the
slopes of these regression lines to differ from
one another. Thus, not only does the biological
value of a protein depend on its concentration
in the diet, but the quantitative relationships
between different proteins depends on the level
at which the proteins are fed.

1793. Role of fat in metabolism under stress
conditions. Hazet M. Fox,* PEaru P. Swan-
SON AND EMERSON Birps.* Nutrition and Dairy
Industry Labs., Iowa Exper. Station, Ames.
Previous reports from this laboratory have

shown that dietary fat is effective in reducing the
catabolism of tissue protein characteristic of rats
fed } of the amount of a protein-free ration (i.e.,
14 calories) that they customarily eat each day.
The protective portion of a commercial cotton-
seed oil of known history appears to have been
identified. The oil was fractionated into several
portions. The distillate designated as Fraction I
contained most of the non-saponifiable materials
in the oil. It proved only 3 as effective as cotton-
seed oil in reducing tissue catabolism when used
in an equivalent amount in the ration. However,
after removal of the non-saponifiable matter, the
residue exerted a protective action equivalent to
that of the original fat. Determination of the
concentrations of oleic and linoleic acids (alkali
isomerization) in the origina! cottonseed oil,
Fraction I, and the distillation residue indicate
that the protective effect may be due to linoleic
acid. The respective amounts of the acid in the
three preparations were 51.5%, 37.8% and 52.4%.
It seems, therefore, that even though the rations
contained essential fatty acid and tocopherols in
amounts ordinarily considered adequate, the
stress condition increased the need for highly un-
saturated fatty acid. (The authors gratefully
acknowledge the analyses made by E. G. Ham-
mond and P. Chandler.)

1794. Preservation of vitamin A in feeds. J.C.
Fritz, F. D. WHarton, Jr.,* R. M. HENtEy*
AND R. B. ScHoEne.* Dawe’s Labs., Chicago,
Til.

Chick studies were used to evaluate rate of loss
of vitamin A and the influence of protective
measures. Criteria used were chick growth and
liver storage. Losses of fat-soluble vitamins from
poultry feeds were shown to be reduced by a)
suitable coating to minimize air contact and by
b) use of antioxidants. Water-soluble coating
agents were satisfactory when feed was stored

552

under conditions of low humidity. They did not
give adequate protection when the feed was stored
under high humidity conditions. Fat-soluble
coating agents were effective in protecting the
fat-soluble vitamins, but care in selection of the
coating agent was necessary to assure that the
coated vitamins were biologically available. High
melting fats used as coating agents, impaired
utilization of vitamin A. Inclusion of either
butylated hydroxyanisole (BHT) or diphenyl-para-
phenylenediamine (DPPD) at the rate of 0.25
lb/ton of feed improved the apparent utilization
of added vitamin A or carotene.

1795. Effect of cocarboxylase on incorporation
of pyruvate into acids of the tricarboxylic
acid cycle by normal and diabetic rat liver.
CHARLES E. FrRoHMAN (introduced by JAMES
M. OrteEN). Dept. of Physiological Chemistry,
Wayne Univ. College of Medicine, Detroit,
Mich.

Previous work has shown that in diabetic rat
liver, acetate does not enter the tricarboxylic acid
cycle in the same manner as in normal liver. C™
from administered acetate appears in lower
concentration in the acids of the cycle in the livers
of rats treated with alloxan. In the present study,
2-C-14 pyruvate was administered to normal rats
and the radioactivities of the cycle acids from liver
were measured 5 min. later. The pyruvate was
incorporated at approximately the same rate as
the acetate. This experiment was then repeated
on alloxan diabetic animals and on alloxanized
rats given cocarboxylase. The results will be
discussed. (Supported by grant No. A-237(c)
from the Natl. Insts. of Health.)

1796. Blood glucose responses to the energy-
yielding nutrients. J. H. Fryer (introduced
by L. A. Maynarp). School of Nutrition, Cornell
Univ., Ithaca, N. Y.

Comparison has been made in normal human
subjects of the effect of ingesting purified carbo-
hydrate, protein or fat on absolute blood glucose
levels gnd upon capillary-venous glucose dif-
ferences. Blood glucose levels remain at or near
fasting level after the administration of protein
in the form of calcium caseinate or fat in the form
of butter. The blood glucose response to an
ingested mix containing 110 gm of glucose and
114 gm of calcium caseinate was significantly less
than the response to a mix of equal volume con-
taining only 110 gm of glucose. This effect may be
partly explained by a reduction in the rate of
gastric emptying produced by the addition of
casein to the mix. Assessment 0” this factor was
made possible by the addition of barium sulfate
to the mixes, enabling gastric emptying times to
be determined by an x-ray method. It was shown

FEDERATION PROCEEDINGS

Volume i§

that approximately 50% of the calcium caseinate
and glucose mix was retained in the stomach at
the end of 2 hr., whilst only about 10% of the mix
containing glucose alone was retained in this
period. Delayed absorption due to a competitive
situation at the intestinal mucosa has also been
postulated. These results demonstrate that blood
glucose responses do not quantitatively reflect
the total caloric intake or the intake of carbo.
hydrate if protein, as casein, is ingested simul-
taneously.

1797. Low-aqueous filter paper electrophore.
sis: applications to phosphatides and bile
acids. JAMES E. GaRvIN (introduced by R. P,
GEYER). Dept. of Nutrition, Harvard School of
Public Health, Boston, Mass.

A method of filter paper electrophoresis employ-
ing solvent mixtures of low water content has been
developed and separations of some phosphatides
as well as some bile acids have been carried out.
A solvent mixture consisting of 47% v/v 2-meth-
oxyethanol, 47% v/v tetrachloroethane, and 6%
v/v water was found suitable for the phosphatides,
Substitution of tetrachloroethylene for the highly
toxic tetrachloroethane permitted only 3% v/v
water and gave less distinct bands. The apparent
pH (measured by glass electrode) was maintained
at about 6.7 by means of a buffer consisting of
benzoic acid (0.05m), potassium benzoate (0.05m),
The migrations of species containing amino
nitrogen were followed with ninhydrin, whereas
the lecithins were followed with the method of
Chargaff (J. Biol. Chem. 175: 67, 1948). Separations
were obtained as follows: phosphatidyl serine
from phosphatidyl ethanolamine, phosphatidyl
serine from lecithin, and phosphatidyl serine
from total rat liver lipid. The phosphatidy] serine
and phosphatidyl ethanolamine were prepared by
the method of Folch (J. Biol. Chem. 146: 35, 1942),
the lecithin by the method of Pangborn (J. Biol.
Chem. 188: 471, 1951). Separation of cholic acid,
synthetic glycocholic acid (BERGsTRoM, Acla
Chem. Scand. 7: 1126, 1953), and synthetic tauro-
cholic acid (NorMAN, Arkiv Kemi 8: 331, 1955)
has been accomplished in a solvent mixture
consisting of 94% v/v 2-methoxyethanol and 6%
v/v water. The buffer and apparent pH were
similar to those described for the phosphatides.

1798. Effect of vitamin D-deficient diets con-
taining various Ca:P ratios on cats. S. N.
GersHorr* AND D. M. Heasvep. Dept. of Nu
trition, Harvard School of Public Health, Boston,
Mass.

Four groups of kittens have been maintained
for more than a year and a half on purified diets
with and without vitamin D and containing either
1% Ca and 1% P or 2% Ca and 0.7% P. Rickets

a, a nn ee ee ee ee ee. ee, en ee ee ee ee ae

_—

olume 1§

aseinate
mach at
the mix
in this
petitive
lso been
at blood
y reflect
f carbo-
1 simul-

ophore-
nd bile
ry R. P.
school of

employ-
has been
phatides
‘ied out,
2-meth-
and 6%
hatides,
e highly
3% v/v
parent
intained
sting of
(0.05m),
amino
whereas
thod of
arations
| serine
yhatidyl
| serine
7] serine
ared by
5, 1942),
J. Biol.
ic acid,
1, Acta
¢ tauro-
1, 1955)
mixture
and 6%
HH were
tides.

ts con-
. SN,
of Nu
Boston,

ntained
ed diets
g either
Rickets

March 1956

produced: by diets with the 1:1 Ca:P ratio was
much more severe than that produced by the high
Ca:P ratio as judged by x-ray, serum alkaline
phosphatase and longevity data. Serum Ca, P,
protein and citric acid and urinary Ca, P, N and
citric acid values have also been obtained. After
about a year on the experimental diets a marked
spontaneous improvement was observed in the
condition of all but one of the rachitic cats. This
probably indicates a very low vitamin D require-
ment in cats 2 yr. or older. Alterations in the ex-
perimental diets obtained by changing their Ca
but not their P levels to produce 0.5:1, 1:1, 2:1
and 3:1 Ca:P ratios, did not result in significant
changes in urinary Ca but produced approxi-
mately 10-fold increases in urinary P as the Ca:P
ratio was lowered from 3:1 to 0.5:1. There were
no significant differences in the urinary Ca, P
excretion of cats receiving and not receiving
vitamin D.

1799. Efficiency of tryptophan as a niacin
precursor. Grace A. GoLpsmiTH, O. NEAL
MILLER AND WALTER G. Unataus.* Depts. of
Medicine and Biochemistry, Tulane Univ., New
Orleans, La.

Fourteen subjects were maintained during 19
experimental periods on controlled diets of low
or moderately low niacin and tryptophan content.
Excretion of niacin and tryptophan metabolites
was determined on the control diet and following
administration of niacinamide (10-15 mg daily)
for one or more test periods and of tryptophan
1-3 gm daily) for comparable test periods. The
amount of tryptophan which appeared to be
equivalent to 1 mg of niacin, as judged by ex-
cretion of niacin metabolites, varied from 31 to
87 mg with an average of 55 mg in the 19 experi-
ments. The efficiency of conversion of tryptophan
to niacin on a molar basis will be discussed.

1800. Basal metabolism of girls in the Great
Lakes region. IstpoR GREENWALD. New York
Univ. College of Medicine, New York City.
In the paper of this title (J. Am. Dietet. Assoc.

30: 986, 1954), Kenyon, Kelly and Macy con-

cluded that the basal metabolism of a group of

girls in Detroit was low. The values were obtained
with the Benedict-Roth apparatus and the ac-
companying chart. Two or three 10-min. periods
were recorded per day, after preliminary practice.

“From among tests which did not agree within

5%, the lowest was recorded.’’ Such values may be

more correct than those obtained in the usual

manner but they are not comparable. Moreover,
as many as 62 determinations were made upon the

same individual, the average number being 10.6.

Even so, some of the median values for Cal/m*/hr.

are higher than the standards that accompany the

AMERICAN INSTITUTE OF NUTRITION

553

instrument or than those given by Boothby,
Berkson and Dunn. They are regularly higher
than the values compiled by Rose (SHERMAN,
Chemistry of Food and Nutrition, 6th ed.). The
figures for Cal/kg/hr. are also frequently higher
than those given by MacLeod and Taylor (Sher-
man, 7th ed.). The oxygen consumption was
calculated from the heat output by using respira-
tory quotients greater than 0.82. If these are
really correct, all the values for heat output should
be increased by about 1% in order to make them
comparable with those in which both oxygen
consumption and carbon dioxide production were
measured. The metabolic rate was not, as claimed,
slightly lower than normal, but, indeed, rather
higher.

1801. Hemin chromoproteins in copper-de-
ficient and iron-deficient swine. C. J.
GuBLER, G. E. CartwriGut anp M. M. Win-
TROBE.* Dept. of Medicine, Univ. of Utah Col-
lege of Medicine, Salt Lake City.
Copper-deficient, like iron-deficient, swine

develop a severe microcytic hypochromic anemia.
In iron-deficient animals the erythrocyte survival
time is normal. Anemia develops because of
decreased hemoglobin synthesis. In _ copper-
deficient anima]s anemia is a result of a shortened
life span of the red blood cells coupled with an
inability of the bone marrow to increase its
synthetic capacity sufficiently to meet the in-
creased demands. In order to study the effect of
these deficiencies on other hemin chromoproteins
the levels of cytochrome c, cytochrome oxidase,
catalase and myoglobin were measured in the
tissues of normal, copper-deficient and iron-
deficient swine. With the exception of cytochrome
oxidase, the concentration of hemin chromo-
proteins in the tissues of iron-deficient pigs was
reduced. A marked increase in cytochrome c
content of the heart and a decrease in catalase
and cytochrome oxidase activity in the liver and
in cytochrome oxidase activity of the heart was
observed in copper deficiency. The myoglobin
content of the leg muscles was unchanged while
the concentration in the heart was decreased by
copper deficiency. The total myoglobin per heart
was normal due to marked cardiac hypertrophy.
These studies indicate that a deficiency of iron
affects the synthesis of myoglobin and the ‘paren-
chymal’ hemin chromoproteins in the same way
as it does hemoglobin. In copper deficiency, a
defect in hemin chromoprotein synthesis only
becomes manifest when the requirement is in-
creased by a shortened life span of the component
or by increased mass of the tissue requiring it. The
behavior of cytochrome oxidase suggests a re-
lationship to copper.

554 FEDERATION PROCEEDINGS

1802. Effects of various minor growth factors
and antibiotics on growth of rats fed a
suboptimal diet. W. KNowiton HAt.t,*
V. P. SyDENSTRICKER AND RoBErtT W. OLIVER.*
Depts. of Biochemistry and Medicine, Med.
College of Georgia, Augusta.

Wistar strain rats when 21 days of age were
placed on a diet consisting of vitamin-free casein,
9 gm, a salt mixture, cottonseed oil and vitamins
A, Bi, Be, Bs, C, D, E and calcium pantothenate
with choline chloride, and starch or sucrose as the
carbohydrate. Increasing the casein level to 20%
gave maximum growth. Adding methionine and
the five other amino acids suboptimal in this diet
to the basal resulted in nearly maximum growth.
The basal diet with methionine, and sucrose as the
carbohydrate, gave } of the maximum growth rate.
Growth on this diet and the other diets containing
inadequate tryptophane levels was improved by
the addition of tryptophane or niacin while growth
on some diets was reduced by suceinylsulfathi-
azole. In the suboptimal diets starch usually re-
sulted in better growth than when sucrose was
used. Aureomycin had little if any beneficial effect
on growth, while addition of penicillin resulted
in a significant increase. PAB and inositol im-
proved growth when added to the basal plus me-
thionine with starch as the carbohydrate but not
when sucrose was used. Vitamin By and folic acid
appeared to have no effect when added to diets in
this experiment.

1803. An amino acid test diet for salmon.
Joun E. HAtver (introduced by Epwin T.
Mertz). Salmon Nutrition Lab., U. S. Fish
and Wildlife Service, Cook, Wash.

An amino acid test diet for chinook salmon (On-
corhynchus tshawytscha) was developed which con-
tained (in %) L-amino acid mix 35, carboxymethy]-
cellulose 5, dextrin 3, corn oil 2.5, minerals 2,
alpha cellulose + vitamins 1.5, cod liver oil 1, and
water 50. The level of each crystalline L-amino
acid in the test diet was (in %) arg-HCl 2.5, his-
HCI1-H.O 1.25, ileu 2, leu 3, lys-HCl 2.5, met 1,
phe 2, thy 1.25, try 0.5, val 2, ala 1.75, asp 2.5, cys
0.25, glu 4, gly 2.5, pro 2.5, ser 1.5, and tyr 2. The
diet was prepared by adding all the dry ingredients
except carboxymethylcellulose to warm water,
blending thoroughly in a mechanical mixer, add-
ing the carboxymethylcellulose and mixing to the
desired consistency. The diet could be prepared
as a floating or sinking diet by altering the speed
of the mixer. The diet was tested for 14 wk. and
comparisons with a complete vitamin test diet
indicated no significant differences in growth or
mortality. By deleting the amino acids one at a
time from this diet, it was possible to classify the
indispensable amino acids for chinook salmon (D.

Volume 1§

C. DeLonea, J. E. Hatver anv E. T. Mertz,
Federation Proc., this issue.)

1804. Water-soluble vitamin requirements of
Chinook salmon. JoHn E. HALVER (introduced
by Epwin T. Mertz). Salmon Nutrition Lab.,
U.S. Fish and Wildlife Service, Cook, Wash.
A vitamin test diet for chinook salmon (On-

corhynchus tshawytscha) was developed which con-
tained (in %) vitamin-free casein supplemented
with pL-methionine and L-tryptophan 55, purified
gelatin 15, corn oil 9, white dextrin 8, alpha cellu-
lose flour 9, and mineral mixture 4. To 100 gm of
this basal diet was added (in mg) thiamin- HCl 6,
riboflavin 20, pyridoxine: HCl 4, nicotinic acid 80,
calcium pantothenate 28, inositol 400, biotin 0.6,
folic acid, 1.5, p-aminobenzoic acid 40, choline 800,
ascorbic acid 200, alpha tocopherol acetate 40,
menadione 4, beta carotene 1.2, activated 7-dehy-
drocholesterol 0.0045, and crystalline vitamin By,
0.009. The diet was prepared by adding the other
dry ingredients to a warm 5% gelatin solution and
blending thoroughly in a mechanical mixer. Prepa-
ration as a floating or sinking diet could be ob-
tained by altering the speed of the mixer. A float-
ing diet containing 25% solids was found optimum
for initial feeding and a slowly sinking diet con-
taining 30% solids was found acceptable for more
advanced feeding. By deleting the purified vita-
mins one at a time from this diet, deficiency syn-
dromes in chinook salmon were observed for thi-
amin, riboflavin, pyridoxine, pantothenic acid,
inositol, biotin, choline and folie acid. The results
with nicotinic acid and vitamin B,2 suggested pos-
sible requirement, but lack of ascorbic acid and
p-aminobenzoic acid did not produce any recog-
nizable deficiency symptoms.

1805. Dietary practices of three samples of
women. LAURA HARPER* AND MARGARET A,
Oxuutson. Foods and Nutrition Dept., Michigan
State Univ., East Lansing.

This study reports dietary intakes of 3 samples
of women over periods from 6 to 20 yr. and through
successive decades as determined from non-con-
secutive 1-day dietary recall records and related
data. Data were collected from a highly trained
group of women, 17-44 yr. old, sample A, and from
2 urban samples, 40-90 yr. old, samples B and C.
Samples B and C consisted of 97 white and 104
negro subjects, respectively. Mean daily caloric
intakes were 2023, 1580 and 1222 calories for sam-
ples A, B, and C. Caloric intakes decreased with
age, except for sample C. The mean gm of protein
for 1000 gm of mixed diet for samples A, B and C
were 36.2, 33.5 and 38.2, respectively. Selected
sample A had chosen a definite food pattern. Al-
though the pattern of sample B was different, both
were rigid. Subjects of sample C were more per-

aie

- at a Se eet, ae

ume 1§

TERTZ,

nts of
oduced
v Lab.,
Vash.
n (On-
-h con-
nented
urified
. cellu-
| gm of
HCl 6,
cid 80,
in 0.6,
ne 800,
ate 40,
'-dehy-
nin Bus
2 other
on and
Prepa-
be ob-
\ float-
timum
at con-
r more
d vita-
'y syn-
or thi-
+ acid,
results
ed pos-
id and
recog-

les of
RET A,
ichigan

amples
hrough
yn-con-
related
trained
id from
and C.
nd 104
calori¢
or sam-
d with
protein
y and C
elected
rn. Al-
it, both
re per-

March 1956

missive in eating practices. All sample A subjects
used the 3-meal-a-day eating pattern. Of samples
B and C 6 and 48%, respectively, ate fewer than
3 meals. The weights of 66, 44, and 24% of samples
A, B and C subjects were considered normal.
Fifty-six, 46, and 22% of samples A, B and C used
vitamin concentrates regularly. Individuals using
vitamin supplements consumed larger amounts of
nutrients from food than did subjects without
supplements.

1806. Resolution of cellulolytic enzymes
from Myrothecium verrucaria. JoHN H. Hasu
AND KENDALL W. Kine (introduced by A. E.
ScHAEFER). Dept. of Biochemistry and Nutrition,
Virginia Polytechnic Inst., Blacksburg.

The complexity of the enzymes in culture fil-
trates of M. verrucaria was investigated by paper
electrophoresis. Dialyzed filtrates, concentrated
200-fold, were applied to Whatman no. | filter
paper, and migration was effected under a poten-
tial gradient of 12/em for 6 hr. in veronal buffer
(ionic strength 0.05, po 8.5). The strips were then
cut into 0.5-em sections, and each section was
assayed for beta-glucosidase and cellulase. Beta-
glucosidase was assayed by determining the 6-
bromo-2-naphthol liberated from 6-bromo-2-naph-
thyl-beta-p-glucopyranoside. Cellulase activity
was measured by the increase in reducing sugars
using a carboxymethyleellulose substrate. Beta-
glucosidase was restricted to three sharp peaks,
but cellulase activity was somewhat diffuse. A
small amount of both enzymes remained at the
origin. Typical electropherograms showed a domi-
nant beta-glucosidase peak migrating 10 em and a
much smaller one at 13 em. The major cellulase
peak moved 6.5 cm but small amounts of activity
were also associated with the two beta-glucosidase
peaks. These data indicate that for the most part
cellulase and beta-glucosidase activity in these
filtrates are dependent on different proteins, and
suggest that several enzymes may contribute to
each function.

1807. Dietary study in a Thai village. Haze.
M. Hauck AND SAOVANEE SupDSANEH.* Food
and Nutrition Dept., Cornell Univ., Ithaca,
N.Y.

As part of a larger investigation on factors in-
volved in cultural change, studies of food intake
and nutritional status were made in Bang Chan,
arice producing village 20 miles from Bangkok,
during 1952-54. For the weighed dietary study, 11
households were chosen, comprising two groups,
with apparently differing nutritive status, the
members of which could be matched approxi-
mately. Conservative estimates of per capita
dietary allowances for the two groups agreed
closely. Each household was visited 4 times in 1

DC aw ana

AMERICAN INSTITUTE OF NUTRITION

595

yr., and all food for 24 hr. weighed before and
after cooking. Nutrient intakes were calculated
from values in tables based largely on analyses
made elsewhere in Southeast Asia. Differences in
calculated per capita nutrient intakes of the two
groups were small but, in general, favored the
group with apparently better nutritive status. For
both groups, rice provided more than 3 of the
calories and over half the protein. Seven house-
holds used polished rice exclusively. About 4 of
the protein was of animal origin, chiefly from fish.
Intake of visible fats and sugars was small. Esti-
mated vitamin A value was largely from vegetable
sources, although the vitamin A value of many
kinds of fish used was unknown. Signs of possi-
ble nutritive deficiency noted, namely tongue
changes, angular stomatitis, changes in skin and
hair and absence of knee and ankle jerk, might
reasonably be associated with apparent deficien-
cies of riboflavin, vitamin A and thiamin. Aside
from short stature and small bones, evidences of
calcium deficiency were not observed, although
estimated calcium intake was low.

1808. Value of further improvement in bread
quality as indicated by rat feeding studies.
E. E. Hawtey. Pediatric Dept., School of Medi-
cine and Dentistry, Univ. of Rochester, Rochester,
Weeks
Comparison of nutritive value of bread made

from formula developed by McCoy and ccllabora-

tors at Cornell with other available wheat breads
has been followed for 4 yr. by rat feeding experi-
ments. ‘Cornell formula’ bread consists of en-
riched flour with 2% wheat germ, 6% full fat soy
and 8% skim milk solids added. This bread—as
well as 20 others—was dried, ground and mixed
with 10% margarine and viosterol. All rats re-
ceived equal amounts of crumbs—the amount the
poorest eaters in the series consumed. Effects thus
reflected qualitative differences between breads.

The dried crumbs were found to be essentially

equal calorically. Water was allowed ad libitum.

Rats, in separate cages, were placed on diet at

weaning. Usual procedure in selection and care

was followed. Comparison was made of: general
appearance, disposition, growth, establishment
and regularity of estrus, reproduction and ability
to produce successive generations. Photographs
supplement data. Superiority of the Cornell for-
mula is in line with improvement in quality of
protein and increase in vitamin-mineral content.

To date 8 generations have been reared on this

improved formula. There is no living 3rd genera-

tion on any standard ‘enriched’ white bread. Eco-
nomic and nutritional value of this improved loaf

—without cost increase or palatability decrease—

deserves consideration, especially in diet of the

aged, children or where food budget is limited.

556

Recognized improvement in health following ‘en-
richment’ demonstrates the importance of bread.

1809. Nutritional studies on acetylated mono-
glycerides. Absorption of acetic acid from
stomach and deposition of lipid-bound,
volatile acids in tissues. Davin C. Hertine,*
Stantey R. Ames,* Norris D. EMBREE* AND
Puitip L. Harris. Research Labs., Distillation
Products Industries. Div. of Eastman Kodak
Co., Rochester, N. Y.

Although large amounts of free, long-chain fatty
acids were found in the lipid recovered from the
stomachs of rats fed saturated acetylated mono-
glycerides (Federation Proc. 14: 173, 1955), only
traces of free acetic acid have been found. Experi-
ments with pylorus-ligated adult rats indicated
that acetic acid is absorbed through the stomach
wall. At the highest level of administration, be-
tween 1.5 and 2.0 mo of acetic acid was absorbed
during a 6-hr. period. Under similar conditions,
about 0.33 mm of glycerol and about 0.32 mm of
monoacetin disappeared. An analytical procedure,
including steam distillation and paper chroma-
tography, was developed for the isolation and
identification of lipid-bound, short-chain, car-
boxylic acids. As little as 0.02 um of the acids can
be detected. Various tissue fats from rats, dogs
and human beings were saponified and analyzed
for water-soluble, steam-distillable acids. Feeding
acetylated monoglycerides to rats at dietary levels
up to 50% increased the lipid-bound, steam-dis-
tillable acids in blood but not in the carcass fat.
The low levels of lipid-bound, steam-distillable
acids which were found in tissue fats from a dog
fed a standard dog-meal were not elevated by
feeding a diet containing 25% acetylated mono-
glycerides. Samples of human blood lipids con-
tained relatively large amounts of water-soluble,
steam-distillable acids.

1810. In vitro hydrolysis and esterification
of vitamin A. Epwarp G. Hien, Henry B.
BriGHT AND J. RoNALD POWELL (introduced
by Harry G. Day). Dept. of Biochemisiry,
Meharry Med. College, Nashville, Tenn.

A study has been made of the in vitro hydrolysis
of vitamin A acetate, palmitate, and natural ester
and the in vitro esterification of vitamin A alcohol.
Tissues from young albino rats (Wistar strain),
maintained on a vitamin A-deficient diet in order
to ensure a low storage of the vitamin, were em-
ployed, the method being substantially the same
as that used by McGugan and Laughland (Arch.
Biochem. 35: 428, 1952). The optimum px for the
hydrolysis of vitamin A acetate was found to be
between 8.5 and 7.9. Very little difference was ob-
served in the effect of dispersing agents (Tweens
20, 40, 60 and 80) on the rate or quantity of hy-

FEDERATION PROCEEDINGS

Volume 16

drolysis of vitamin A acetate by liver homoge-
nates. The rate of hydrolysis of vitamin A acetate
was greatest in the liver, intermediate in the
blood, and least in the kidneys. The hydrolysis of
vitamin A natural ester and palmitate by these
tissue homogenates was practically nil. On the
other hand, the in vitro esterification of vitamin A
alcohol was found to proceed well in both the in-
testine and kidneys with the blood and liver ex-
hibiting very little esterification. It is suggested
that during the metabolism of vitamin A the natu-
ral ester is converted to shorter chain esters before
final hydrolysis, that the liver is the chief site of
these processes, and that the kidneys are the chief
site of postabsorptive esterification. These ob-
servations are in accord with the view advanced
by High and Wilson (Arch. Biochem. & Biophys,
In press) that kidney vitamin A originates from
liver vitamin A alcohol and that esterification of
the alcohol takes place in the kidneys.

1811. Effect of choline chloride on response of
chicks to a transplantable tumor (RPL-12),
C. H. H1iu anp H. W. Garren (introduced by
G. H. Wise). Dept. of Poultry Science, North
Carolina State College, Raleigh.

Studies have been undertaken to determine the
effect of dietary variations on the resistance of
chicks to the transplantable tumor RPL-12. One
of the first organs that is affected by metastases
of this tumor is the liver. Therefore, experiments
have been conducted to determine the effect of
some lipotropic agents on the resistance of chicks
to this tumor. The experimental diets were based
on soybean meal and corn and contained all the
known required nutrients in adequate amounts,
including 1300 mg choline/lb. Chicks were ob-
tained from a commercial hatchery and placed on
the experimental diet at 1 day of age. At 6 days
they were inoculated in the pectoral muscle with
either 500 ugm of fresh tumor or 10 mg of frozen
tumor. Two weeks after inoculation all the chicks
were killed and examined carefully for the pres-
ence of the tumor. In three experiments the per-
centage of chicks, fed the basal diet, positive for
tumor was 25, 44 and 79. When 1.4% choline chlo-
ride was added to the feed, the corresponding per-
centages were 16, 23 and 52 respectively. Neither
betaine hydrate (1.4%) nor pu-methionine (2%)
caused a decrease in the percentage of chicks posi-
tive for tumor.

1812. Niacin - tryptophan requirements of
man. M. K. Horwirr. Dept. of Biological
Chemistry, Univ. of Illinois College of Medt-
cine, Chicago, and Elgin State Hosp., Elgin, I
Comparisons of data obtained during Elgin

studies on niacin-tryptophan requirements with

reports on the induction of pellagra in man indi-

ee ee ee ee ee ee a ee oe a ne

Qn eo = + ©) e

—
—

lume 16

jomoge-
acetate
in the
lysis of
yy these
On the
famin A
. the in-
iver ex-
iggested
he natu-
's before
f site of
he chief
ese ob-
dvanced
Biophys.
tes from
ation of

ponse of
(PL -12),
luced by
e, North

mine the
tance of
-12. One
-tastases
eriments
effect of
of chicks
re based
1 all the
:mounts,
vere ob-
laced on
t 6 days
scle with
of frozen
he chicks
the pres-
the per-
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. Neither
ine (2%)

icks posi-

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Elgin, Ill
ng Elgin
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March 1956

cate that, approximately 200 mg of tryptophan
plus 5 mg of niacin is a borderline intake. It is
now generally accepted that tryptophan can sub-
stitute for niacin and an equivalence of 60 mg of
tryptophan for 1 mg of niacin has been tentatively
suggested. If data available from Goldberger’s
classical experiments are correct, then one must
explain why his diets, which provided 6.7 mg nia-
cin and 330 mg of tryptophan (in 3000 Cal.) pro-
ced pellagra, and why the Elgin experimental
diets which provided as little as 5.2 mg of niacin
and 238 mg of tryptophan (in 2070 Cal.) did not.
The explanation may lie in differences in the ca-
loric intake, as the Goldberger diet provided only
4.1, whereas the Elgin diet provided 4.4 niacin
equivalents per 1000 Cal. That a minimum require-
ment for niacin-tryptophan may exist which may
not be related to caloric intake, has been sug-
gested in the Tulane (Goldsmith) studies in which
symptoms of niacin deficiency were obtained on
diets which provided as much as 8.7 niacin equiva-
lents in 1800 Cal. Accordingly, 8.8 niacin equiva-
lents is being suggested as the minimal amount
needed to prevent pellagra in the adult on a diet
which provides 2000 Cal. or less, plus 0.44 niacin
equivalents for each additional 100 Cal. above
2000. The 2000-Cal. figure was chosen because it is
close to the average energy expenditure for an
inactive adult. Obviously, adjustments would
have to be made for the very small, inactive adult.
The niacin equivalents/1000 Cal. (niacin ratio) for
representative foods have been estimated.

1813. Vitamin B, deficiency in rabbits. E. L.
Hove anp J. F. Hernpon.* Dept. of Animal
Husbandry and Nutrition, Alabama Polytechnic
Inst., Auburn.

Weanling rabbits required more than 20 ug of
pyridoxine/day (0.5 mg/kg diet) for maximum
growth of 17 gm daily on a synthetic type diet
with 25% casein. The average growth rate of 18
rabbits on the deficient diet was 3.4 gm daily; 8
died between 63 and 150 days. The vitamin Bg
content of the livers was 1.41, 3.00, and 6.84 ug/gm
for the deficient, 20 wg daily, and 200 ug daily
treatments respectively. The vitamin B, content
of soft feces (dry) was 1.10 ug/gm for deficient
animals and 4.21 wg/gm for controls; the hard
feces contained 0.37 and 0.78 ug/gm respectively.
Gross symptoms of the deficiency included en-
crustation of the eyelids and nose, dermatitis of
paws, marked scale formation on ears and fore-
legs, anemia, xanthurenic acid excretion, and pro-
longed blood clotting time. Various neurological
abnormalities were shown. Extreme trembling and
incoordination as well as the typical convulsion
pattern familiar to other species were frequent.
in addition, 4 rabbits displayed sudden collapse
of the hind quarters with paralysis; 5 others had

AMERICAN INSTITUTE OF NUTRITION

557

neck, head and forequarter involvement. High
urinary creatine was noted only in moribund ani-
mals. The vitamin E requirement of rabbits was.
not increased in the vitamin Bg deficiency. Symp-
toms of the deficiency were not aggravated by
omission of all fat from the diet. (Aided by Re-
search Grant B-430 from the Natl. Inst. of Neuro-
logical Diseases and Blindness.)

1814. Deficiencies of certain B vitamins and
absorption of vitamin B,.. JEnc M. Hsvu,*
Bacon F. Cuow, Kunio Oxupa* aNnp ERNEs-
TINE B. McCoutium. Dept. of Biochemistry,
School of Hygiene and Public Health, Johns
Hopkins Univ., Baltimore, Md.

Several experiments were carried out to deter-
mine whether deficiency of certain B vitamins in-
fluences the absorption of B,2. Animals were kept
on a basal casein-sucrose diet supplemented with
all known vitamins, except the one deficiency of
which was to be produced. Half of the animals
(control) were given this vitamin intraperito-
neally. After symptoms of deficiency developed,
rats in both groups were given orally a test dose of
50 my of Biz labeled with (Co). Urinary and fecal
samples were separately collected for 5 days. The
animals were killed and their kidneys, liver and
gastrointestinal tracts were removed for radio-
activity measurement with a scintillating counter.
The results of one experiment are tabulated below.
These indicate that pyridoxine deficiency in adult
female rats produces increased radioactivity in
feces but not in urine, and suggest the impairment
of absorption of B,.. The phenomenon is likewise
reflected in differences in the content of radio-
activity in livers and kidneys, but not in the gas-
trointestinal tracts. However, absorption of Biz
is not affected by deficiencies in thiamine, ribo-
flavin or pantothenic acid.

Rapioactivity (% ORAL DOSE)

Feces Urine Liver Kidney Tact
Be-deficient

59.8 1.22 6.2 5.9 10.1

+3.2 +0.12 +0.31 +0.49 +1.2
Be-treated

42.3 1.92 9.2 10.5 9.1

+2.7 +.45 +0.45 +0.05 +1.0

1815. Differences in the metabolism of glucose
and cellobiose by a cellulolytic bacterium.
Frank H. HutcnHer anp Kenpati W. Kine
(introduced by R. W. ENGEL). Dept. of Bio-
chemistry and Nutrition, Virginia Polytechnic
Inst., Blacksburg.

Utilization of cellobiose in preference to glucose
has been demonstrated in a cellulose-decomposing
organism tentatively identified as Cellvibrio ful-

558

vum. The metabolic basis for this discrepancy in
monosaccharide and disaccharide metabolism has
been sought. Cellobiose was the only sugar de-
tected in filtrates of cultures growing actively on
cellulose as sole energy source. Growth and res-
piration data indicate no adaptation to glucose
because cellobiose-grown cells respire glucose im-
mediately at a constant rate, and repeated sub-
culturing in glucose did not enhance growth.
Rapid respiration of glucose by resting cells also
indicates that limited glucose permeability is not
the cause of preferential utilization of cellobiose.
The oxidation of glucose can apparently be cou-
pled to energy-requiring processes since differen-
tial phosphate analyses of cellobiose-grown cells
respiring glucose show an accumulation of organic
phosphate. To some extent the pathways of me-
tabolism of the two sugars appear to differ, since
respiration rates of resting cells oxidizing equiva-
lent concentrations of glucose, cellobiose and a
mixture of the two sugars were in the approximate
ratio of 100:110:140 respectively. Growth rates on
these same substrates were in the same order. The
pH optima for growth differ for the two sugars.
‘The data suggest that in normal utilization of cel-
lulose, cellobiose is the predominant extracellular
hydrolytic product and that the disaccharide is
not converted quantitatively to either glucose or
its immediate phosphorylation products in being
metabolized.

1816. Anti-carnitine effect of y-butyrobetaine
on the development of the chick embryo.
Tosuio Itro* anp G. FRAENKEL. Dept. of En-
tomology, Univ. of Illinois, Urbana.
Carnitine is known as a growth factor for the

mealworm, Tenebrio molitor, and y-butyrobetaine

was found to compete for carnitine (BHaTtTa-

cHaryya et al. Arch. Biochem. Biophys. 54: 424,

1955). The effects of these two substances on the

in vitro development of chick blastoderms were

examined, using Spratt’s method (1947-49). In

most instances, the culture media contained 1.25 x

10-* m p-glucose, the minimum doses for the blasto-

derm to,continue development. Embryos of defini-

tive streak stage or head-process stage explanted
to media containing a 1 X 107 to 3 X 10°? molar
concentration of y-butyrobetaine undergo degen-
eration, the course of which is not identical with
that in the absence of glucose. In the presence of
1 X 10" or 5 X 10°? Mof y-butyrobetaine, the ex-
plants undergo slight development during the
first day and sometimes head-fold or heart are
formed, and degenerate during the next 20 hr. In
the concentration of 3 X 10-2 m, head-fold, heart
and somites are formed in almost all embryos, but
after the first 20 hr. degeneration occurs, espe-
cially in the node and head-fold. If L-carnitine
(2 X 10-* m) is added to media containing 3 X

FEDERATION PROCEEDINGS

Volume 1§

10-2 m of y-butyrobetaine, most of the blastoderms
continue to develop even after 2 days and heart
beat is observed in some of them. The recovery
action by L-carnitine is doubtful with higher con-
centrations of y-butyrobetaine. The embryos of
one-somite to four-somites stage are less affected
by y-butyrobetaine and the antagonistic effect of
L-carnitine is not clear. The results suggest that
carnitine plays an important role in early chick
development.

1817. Growth response of rats on bread mix-
tures containing non-fat milk solids with
and without lysine supplementation. JANE
K. JAHNKE* AND CeEcILIA Scuuck. Dept. of
Foods and Nutrition, Purdue Univ., Lafayette,
Ind.

Growth studies using experimental rats indicate
that lysine is the most ‘limiting’ amino acid in
wheat flour. The addition of non-fat milk solids
to bread formulas increases the lysine content of
bread, but at the levels currently in use lysine
remains quite low. The proposal that lysine be
added as an adjunct to the present enrichment
standards for bread suggests a need for investi-
gating the extent to which nutritional advantages
accrue. In this study the lysine content of the diet
fed young rats was varied by using bread formulas
containing 3, 6 and 12% non-fat milk solids and
formulas with like levels of non-fat milk solids
plus 0.25% lysine. The level of lysine supplementa-
tion was based upon the proposed enrichment level
for bread. The protein content of the diets ranged
from approximately 10 to 13%. The rats were
maintained on the diets for a period of 6-8 wk. As
the levels of non-fat milk solids in the diet were
increased the rats not only showed greater weight
increments but both food efficiency (gain/100 gm
food consumed) and protein efficiency (gain/gm
protein consumed) were improved. Supplemation
with lysine increased weight gains and food and
protein efficiency. The weight gains and food effi-
ciency with 12% milk solids fell in between the
gains with 3% and 6% milk solids plus lysine, but
the protein efficiency was greater for the two
latter diets.

1818. Effect of vitamin injections on survival
of chicks with a high mortality syndrome.
Leo S. JENSEN,* JoHN ALLRED,* Ramon Fry*
AND JAMES McGinnis. Dept. of Poultry Science,
State College of Washington, Pullman.
During a series of nutritional studies it was

noted that chicks from a particular pullet breeder

flock exhibited an abnormally high early mortality.

From 5 to 25% of the chicks died during the first

2 wk. after hatching. High mortality occurred

even though both breeders and chicks were fed

practical rations believed to be adequ ‘te in all

the

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occurred
were fed
te in all

March 1956

nutrients. The mortality has been tentatively at-
tributed to avian encephalomyelitis. In an at-
tempt to reduce mortality, a high level antibiotic
was fed and injections of B-complex vitamins and
penicillin were made. A level of 100 gm terramy-
cin/ton of feed did not reduce mortality. The sub-
cutaneous injection of penicillin plus certain B-
complex vitamins reduced mortality to about 1%.
In another experiment chicks injected with dis-
tilled water, B-complex vitamins, penicillin, and
a combination of penicillin with B-complex vita-
mins had 17, 0, 13 and 2% mortality, respectively.
The chicks receiving vitamins were each given 250
ug thiamine HCl, 250 ug pyridoxine HCl, 500 ug
riboflavin, 500 ug Ca pantothenate, 25 ug folacin,
5ug biotin and 5 wg vitamin By in 0.1 ml of dis-
tilled water. The results also suggest that early
growth of chicks injected with vitamins was in-
creased. In further studies on the problem, the
breeder flock was divided, with } receiving a diet
much higher in vitamins. The first hatch suggested
an improvement in hatchability of fertile eggs for
the group receiving additional vitamins.

1819. Effect of previous calcium intake of
four young men on amount retained from
a low calcium diet. Frances A. JOHNSTON,
CAROLYN TREDWELL AND EvizaBetH Dtao.*
New York State College of Home Economics,
Cornell Univ., Ithaca.

Four college men who had been living on diets
estimated to contain approximately 2000, 1500, 750
and 450 mg of calcium a day, according to a week’s
dietary record, were placed on a diet containing
slightly less than 200 mg/day. The study extended
over five 4-day collection periods, the first of
which served for adjustment. The first subject ex-
creted in feces and urine a mean of 218 mg of cal-
ctium/day more than was contained in his food.
The second subject started with a negative bal-
ance of 127 mg/day and then lost less during each
successive period until during the last 4 days he
lost only 35 mg/day. The third subject, for whom
only one urinary value was obtained, appeared to
be about in equilibrium. The fourth subject re-
tained 92 mg/day. While this indicates that the
previous diet exerts a control over the amount of
calcium retained, the number of subjects is too
few for the drawing of a valid conclusion. More
tases will be studied.

1820. Penicillamine (beta-mercaptovaline)
and growth of chicks. Tuomas H. JUKEs.
Research Div., American Cyanamid Co., Lederle
labs., Pearl River, N. Y.

A growth-depressing effect was noted when L-
penicillamine was added to a purified diet for
thicks at the rate of 3 gm/kg of diet. In contrast
to the corresponding effect with rats (WiLson, J.

AMERICAN INSTITUTE OF NUTRITION

509

E. anv V. DUVIGNEAUD, Science 107: 653, 1948) the
growth depression was not reversed by ethanol-
amine. A growth-promoting effect in chicks was
produced by adding either penicillin or p-penicil-
lamine to the diet; 0.5 mg of procaine penicillin
produced a somewhat greater growth response
than did 10 mg of p-penicillamine/kg of diet. The
diet contained 0.4% cystine, 20% casein and no
inhibitory effect was found for 15 penicillin-sensi-
tive microorganisms when p-penicillamine was
added to agar plates at levels up to 50 ug/ml and
p-penicillamine failed to inhibit the growth of
Clostridium welchii in tube cultures at levels up
to 0.1 mg/ml.

1821. Nutritional quality of food purchases
of urban families. Louis—E KELLEY,* Mar-
GARET A. OHLSON AND GERALD G. QUACKEN-
BUSH.* Depts. of Foods and Nutrition and Agric.
Economics, Michigan State Univ., East Lansing.
Food purchases for 1 yr. of 146 families living in

an industrial area of Michigan during 1953 were
evaluated for calories and 8 nutrients using stand-
ards, outlined by the Food and Nutrition Board of
the National Research Council. Thirty-eight fami-
lies, including 109 adults and 32 children, reported
food purchases with 100% or more of calories and
recommended nutrients available. Sixty-eight
families with 137 adults and 99 children had 80%
or more of calories and recommended nutrients
available. Forty families with 80 adults and 32
children had less than 80% of one or more recom-
mended nutrients available. The percentages of
total available calories derived from protein, fat
and carbohydrate for these three groups of fami-
lies were similar:

% Recommended Source of Cals., %
Prot. Fat CHO

Nutrients
100 or more 12.8 41.1 46.1
80 or more 12.5 41.3 46.2
Less than 80 12.4 42.0 45.6

Protein sources for 3 groups of families differed
somewhat :

Source of Protein, %

% Recommended Milk Meat, ruits,
Nutrients products eggs Cereals veg.
100 or more 26 49 17 8
80 or more 28 39 19 14
Less than 80 22 43 21 14

1822. Effect of dietary lysine on tyrosinase
activity in feather pulp of turkey poults.
F. H. Kratzer anp Pran Voura.* Dept. of
Poultry Husbandry, Univ. of California, Davis.
Bronze turkey poults which were fed a ration

deficient in lysine failed to deposit dark pigment

in their feathers. Feather pulp from normal poults
contained tyrosinase which was capable of form-

560

ing melanin from tyrosine after 5 days’ incubation.
Pulp from poults deficient in lysine contained very
little tyrosinase activity. The same difference in
activity was noted when DOPA was used as a sub-
strate instead of tyrosine, except that the time of
the reaction was greatly reduced. The results indi-
cate that the defect in pigment formation seen in
lysine deficient poults is caused by a reduced en-
zyme activity rather than a lack of substrate for
melanin formation.

1823. Sulfur metabolism in baby pigs. R.
Kutwicu,* L. Strueiia* anp P. B. Pearson.
Animal and Poultry Husbandry Research Branch,
USDA, Beltsville, Md.

Studies have been made on the absorption, ex-
cretion and deposition of S**. A single dose of
about 1 me of S*-labeled sulfate together with
0.2 mg of carrier sodium sulfate was administered
by stomach tube to each of three 17-day-old gilts
that were litter mates. They were individually
caged and fed pasteurized whole cows’ milk 4
times daily for 4 days preceding dosing and during
the 4-day collection period which followed. About
7% of the dose was excreted in the urine during the
first 4 hr., a total of 37% during the first 24 hr. and
about 62% during the entire 4-day collection pe-
riod. About 92% of the serum S** appeared in the
fraction insoluble in 90% ethanol. The tissues
analyzed for radiosulfur content, listed in order
of diminishing S** concentration, were: ear car-
tilage, red bone marrow, aorta, femur shaft, skin,
serum, liver, brain, kidney, heart and gastrocne-
mius muscle. Almost half of the radiosulfur in a
hydrolyzate of gastrointestinal tract contents was
present in the cystine and methionine fractions
separated by chromatographic methods. Data will
be presented on the distribution in tissues and
excreta of various sulfur-containing compounds.

1824. Nutritional and genetic determinants
in acute disseminated encephalomyelitis
in mice. JOHANNA M. LeEE,* Howarp A.
ScHNEIDER AND PETER K. Ouitsky.* Rockefeller
Inst. for Med. Research, New York City.
Genetig. studies have led to the identification of

a mouse genotype (BSVS) strain which is 100%

susceptible to acute disseminated encephalomye-

litis (ADE). This malady was produced by serial
intracutaneous injection of homologous mouse-
brain proteolipid admixed with a Freund-type ad-
juvant. The 100% susceptibility of the BSVS
mouse when reared on a diet of fox chow-bread and
milk was reduced by 90% when weanlings were fed

a simplified synthetic diet. This latter diet con-

tained no supplements of folic acid, Biz or biotin.

Single supplementation of these vitamins, in lib-

eral amounts, resulted in a restoration of a vari-

able susceptibility. Supplementation with all three
led to the maximal restoration of susceptibility

FEDERATION PROCEEDINGS

Volume 16

and approached that of control mice fed the afore-
mentioned laboratory stock diet.

1825. Importance of arginine and methionine
for growth and fur development of mink on
purified diets. Witt1AM L. LEoscHKE* anp
Conrap A. ExvenJEemM. Biochemistry Dept.,
Univ. of Wisconsin, Madison.

Svidence available from studies with mink on
purified diets has indicated that the mink, in addi-
tion to the known crystalline vitamins, require 2
unknown factors present in liver and a 38rd un-
known factor present in hog intestinal mucosa for
growth and survival. One of the unknown factors
present in liver has been designated the residue
factor—present in the insoluble residue from 60%
ethanol extraction of liver. The results of present
studies indicate that arginine and methionine may
replace the residue factor required by the mink.
Mink with the characteristic symptoms of the resi-
due factor deficiency, loss of weight, depigmented
fur of poor quality and anorexia, have responded
to the addition of 0.5% u-arginine and 0.25% pbt-
methionine to the diet. An immediate recovery of
appetite was noted with subsequent weight gain.
Mink kits placed on the purified diet supplemented
with arginine and methionine have size and fur
quality comparable to that of mink raised on
ranch diets containing horsemeat, liver, fish and
commercial mink cereal mixtures.

1826. Effect of nutritional supplements on
production of ‘Myleran’ cataracts in rats.
Amos E. Licut, Cyrit Sotomon anp E. J.
DEBEER (introduced by R. J. Buock). Wellcome
Research Labs., Tuckahoe, N. Y. and The French
Hosp., New York City.

Cataracts have been produced in albino rats by
adding from 7.5 to 20 mg of 1,4-dimethanesulfon-
oxybutane, ‘Myleran,’ to each kg of Fox Chow diet
corresponding to intakes of 350 y and 1600 y/kg
body weight daily. Up to the present time such
cataracts have not been found in any other species,
including humans. This drug, in much lower dos-
age levels, is now in common usage for the treat-
ment of chronic myelocytic leukemia. Various
dietary supplements, including vitamins, amino
acids, fats and other adjuncts reported to be nec-
essary in the metabolism of lens tissue, have been
tested for their ability to prevent cataract devel-
opment. Of the materials used, the fats alone were
found to have a pronounced capacity to inhibit
opacity formation and retard other toxicity symp-
toms. For example, a diet containing 10% cod liver
oil prevented cataract formation even though the
‘Myleran’ intake was 870 y/kg body weight/day.
Amounts of vitamins A and D equivalent to those
contained in the oil had no effect in preventing
toxic symptoms. When certain dietary levels of
galactose and ‘Myleran’ were combined in the food

> 2068 a tee. ae a ee... ee i a es a

is :

E XUM

ume 16

afore-

jonine
ink on
* AND

Dept.,

ink on
n addi-
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rd un-
osa for
factors
‘esidue
m 60%
resent
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» mink,
he resi-
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5% DL-
very of
it gain.
mented
und fur
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ish and

nts on
n rats.
» E. J.
“ellcome
French

rats by
»sulfon-
ow diet
00 y/kg
ne such
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ver dos-
e treat-
Various
, amino
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, inhibit
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to those
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evels of
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March 19€6

it appeared that their actions in causing cataracts
were additive, thereby suggesting that these two
substances have certain actions in common. It is
predicted that ‘Myleran’ may prove to be a useful
tool in the study of cataract formation.

1827. Changes in niacin metabolism of rats
produced by ovariectomy and administra-
tion of sex hormones. Mary E. Losxrn,*
Beryt S. BoucHarp* AND ANNE W. WERTZ.
Nutrition Research Lab., School of Home Eco-
nomics, Univ. of Massachusetts, Amherst.

In a previous report (J. Nutrition 46: 335, 1952)
the theory was offered that increases during preg-
nancy in the urinary excretion of N!-methylnico-
tinamide (MNA) and the acid-hydrolyzable metab-
olites of nicotinic acid (NA) might be attributed
to changes in endocrine functions which occur
during pregnancy. In this investigation changes in
hormonal activity were produced by ovariectomy
and administration of ovarian hormones and tes-
tosterone propionate. In adult rats excreting 226
ug of MNA and 133 wg of NA per 24 hr., ovariec-
tomy resulted in decreases of 33 and 23%, respec-
tively. Administration of a combination of 4 mg
progesterone and 0.5 ug estrone for 10 consecutive
days induced marked increases in the excretion of
MNA and NA, which started on the 3rd day in
ovariectomized rats and on the 6th to 8th day in
intact rats, and reached maximum values on the
8th to 15th day after the first injection. The ovari-
ectomized rats’ maximum excretions of MNA and
NA exceeded pre-injection excretions by 134 and
98%, respectively, and the pre-operative excre-
tions by 57 and 48%, respectively. The intact rats’
maximum increases in MNA and NA excretions
were 61 and 45%, respectively. Some similarity
was observed in the response of niacin metabolism
to pregnancy and to ovarian hormonal treatment.
Decreases in MNA and NA excretions induced by
testosterone propionate administration were of
the same order of magnitude for intact and ovari-
ectomized rats.

1828. Quantitative utilization of tryptophan
from foods by the rat. C. H. LusHspoveu,*
THELMA PorTER AND B. 8. ScHWEIGERT. Comm.
on Home Economics and Food Research Inst.,
Dept. of Biochemistry and American Meat Inst.
Fndn., Univ. of Chicago, Chicago, Ill.

This study was undertaken to determine how
much of the tryptophan present in several foods
is actually used to support the physiological func-
tion of growth in the male weanling rat. The diet
was designed to be adequate in all known required
nutrients except tryptophan, so that the limiting
amounts of tryptophan supplied were required
only for protein synthesis. The basal ration pro-
vided 38 mg tryptophan and 2 mg niacin/100 gm.
The growth response of animals receiving graded

AMERICAN INSTITUTE OF NUTRITION

561

supplements of L-tryptophan was compared with
that obtained when fresh or cooked meats, cereals,
legumes or skim milk were fed to provide limiting
levels of tryptophan supplementation. Prelimi-
nary tests indicated that foods added to the basal
ration to provide an additional 40 mg tryptophan/
100 gm were satisfactory for the utilization tests.
Tryptophan content of the foods studied was as-
sayed both microbiologically and chemically and
the results were in good agreement. Tryptophan
utilization results were: meats, 68-94%; peas and
soybean meal, 75-104%; skim milk, 85%; oats,
107%; and unheated soybean meal, 152%. For cer-
tain foods, tests were also run with food supple-
ments providing an additional 80 mg tryptophan/
100 gm ration. Higher apparent utilization was
observed, particularly with rolled oats and heated
soybean meal, as well as with the unheated soy-
bean meal fed at the 40 mg level. The reasons fer
discrepancies have not as yet been ascertained
Fecal tryptophan is now being determined to pro-
vide data on tryptophan digestibility from these
foods.

1829. Nitrogen retention and riboflavin me-
tabolism in human subjects fed low intakes
of the vitamin. Rutu N. Lutz,* Marittyn
B. Dersy,* Berry M. Ernset,* Zarpa H.
Prerce* anp H. H. Wruutams. Depts. of Bio-
chemistry and Nutrition and Food and Nutrition
and the School of Nutrition, Cornell Univ.,
Ithaca, N. Y.

For 42 days, 3 women and 9 men each consumed

a diet containing from 50 to 66% of the recom-

mended allowance for riboflavin. For the following

32 days each subject received between 35 and 40%

of this allowance. The diet contained approxi-

mately 80 gm of protein daily throughout the
study. The total riboflavin content of the erythro-
cytes and the free and total riboflavin content of
the serum of each subject were determined weekly.

Nitrogen retention and riboflavin excretion were

determined for 6-day periods in the 3rd, 7th and

final weeks. Average initial values for total ribo-
flavin content of the erythrocytes and free and

total riboflavin content of the sera were 22.4 (S.E.

2.26), 1.38 (0.279), and 3.85 (0.262) mg % respec-

tively. At the end of the study they were 14.6

(0.52), 0.49 (0.136), and 2.63 (0.157) mg %. There

was a highly significant decrease for all subjects

in riboflavin content of erythrocytes and sera,
although the rate of decrease varied from indi-
vidual to individual. No over-all relationship of
nitrogen retention to erythrocyte or serum levels
of riboflavin existed, although there was some
indication that nitrogen retention may be directly
related to serum riboflavin levels in subjects that
are partially depleted of the vitamin. Considering
all subjects over the entire study, riboflavin ex-

562

cretion and nitrogen retention were inversely re-
lated. (Contribution from Northeastern Nutri-
tional Status Project, NE-16.)

1830. Supplementary value of millet for low-
casein, raw corn diets. AURORA 8S. ManGay,*
WituraMm N. PEarson* AnD WILLIAM J. DaRBy.
Div. of Nutrition, Depts. of Biochemistry and
Medicine, Vanderbilt Univ. School of Medicine,
Nashville, Tenn.

In countries with regions of endemic pellagra
the consumption of millet is associated with a
virtual absence of pellagra. Therefore, the effect
of millet supplementation upon a maize-contain-
ing diet has been studied. The millet used was a
local variety of Setaria italica and assayed 10.86%
protein, .22% tryptophane, and 1.23 mg % niacin.
Groups of 6 weanling Sprague-Dawley rats were
fed a niacin-free diet containing 9% casein + 40%
ground whole corn. The addition of 5%, 10%, 20%
and 40% millet to such a diet (replacing an equiva-
lent amount of sucrose) enhanced growth well
above that obtained when the amount of niacin
found in each millet supplement was added. Maxi-
mum enhancement of growth was produced by
levels of 20% or 40% millet. Inclusion of the tryp-
tophane equivalent alone of each millet supple-
ment produced growth equal to that supported by
the respective level of millet supplement alone.
Inclusion of both the niacin and tryptophane con-
tained in the respective levels of millet produced
no better growth than did tryptophane alone.
These results indicate that the supplementary
value of millet in the corn diet is due primarily to
its tryptophane content. (Supported in part by «
grant from the E. I. du Pont de Nemours Co.)

1831. Xanthomatosis and atherosclerosis pro-
in a Rhesus monkey. GEORGE V.
MANN STEPHEN B. Anprus.* Dept. of
Nutrition, Harvard School of Public Health,

Boston, Mass.

An adult-rhesus monkey was fed diets comprised
of casein or soya protein, dried egg yolk, minerals
and vitamins for a period of 40 months. These diets
contained large amounts of cholesterol. The ani-
mal’s serum lipid levels were increased within a
few weeks and after 6 months the serum choles-
terol level, which originally was 175 mg %, was
900-1600 mg %. The serum £-lipoprotein levels of
the S; 0-35 band were also much increased, but the
serum did not become lactescent. The animal ap-
peared vigorous and healthy throughout the ex-
periment. Cysteine supplements did not affect the
serum lipids. After the animal had been hyper-
lipemic for about 30 months, nodules appeared on
the limbs, especially at points of trauma over the
elbows, hands, soles and ischial tuberosities. There
were no xanthelasma. The electrocardiogram was
not changed and there were no signs of angina

duced
AND

FEDERATION PROCEEDINGS

Volume 16

pectoris. When the monkey was autopsied, exten-
sive atherosclerotic lesions were found in the aorta
and all its major branches, including the coro-
naries, the cerebral vessels, the splanchnic arteries
and the major arteries of the limbs. The vascular,
tendinous and cutaneous nodules resembled the
human lesions in microscopic structure. There
were no visceral accumulations of lipids.

1832. Toxicity of ethionine as influenced by
methionine and by pregnancy. M. ELIzABETH
MarsH, ELeanor S. Irvine,* L. D. GREEN-
BERG* AND J. F. Rinewart.*! Dept. of Pathol-
ogy, Univ. of California School of Medicine,
San Francisco.

The influence of pL-methionine and pregnancy
on the toxic effects of subcutaneous injections of
pL-ethionine was studied in the Long-Evans rat.
The amounts of ethionine employed were 10, 15,
or 20 mg/day. for 9 days. In rats receiving both
methionine and ethionine, the substances were
given in equal amounts. There was considerable
individual variation in toxicity as judged by ano-
rexia, weight loss and pancreatic acinar cell dam-
age. In the nonpregnant rats, 10 mg of ethionine
produced some anorexia in most animals but only
occasional weight loss. Pancreatic damage was
absent or mild. Twenty mg produced a slightly
greater effect on appetite and weight but a marked
increase in the severity of pancreatic damage.
The simultaneous injection of methionine with
ethionine to pair-fed rats reduced, but did not
eliminate, the weight losses. There was only a
slight alleviation of pancreatic damage at both
levels. In rats injected with 10, 15, or 20 mg of
ethionine from the 14th day of pregnancy, the
weight gains decreased with increasing doses. This
parallelism was not reflected, however, in the in-
cidence or degree of anorexia. The stress of preg-
nancy was particularly evident in the pancreatic
damage. Ten mg caused as much damage as did 20
mg in the nonpregnant animals. Fifteen and 20 mg
produced severe damage in the majority of these
rats. In pair-fed pregnant rats receiving both
ethionine and methionine, the damage to the pan-
creas was still severe although there was some in-
crease in the average weight gains. (Supported in
part by a grant from the American Cancer Society
and from the Natl. Vitamin Fndn.)

1833. Hemoglobinuria induced in the vita-
min E-deficient rat by massive injections
of water-soluble vitamin K. WitBuR Marv-
sicH,* ELMER DE Ritrer* AND Saut H. Rvusin.
Nutrition Lab., Hoffmann-La Roche, Nutley, N.J.
Hemoglobinuria in vitamin E-deficient rats in-

jected intramuscularly with massive doses of the

tetrasodium salt of the diphosphoric acid ester of

1 Deceased.

ime 15

exten-
> aorta

coro-
rteries
scular,
ed the
There

ed by
ABETH
'REEN-
Pathol-
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snancy
ions of
ns rat.
10, 15,
g both
s were
lerable
»y ano-
1 dam-
1ionine
it only
ve was
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amage.
e with
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mg of
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he pan-
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e vita-
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Marv-
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rats in-
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ester of

March 1956

2-methy4-1,4-naphtho-hydroquinone (J) was re-
ported by Moore and Sharman (Lancet 1; 819,
1955). With adequate vitamin E in the same diet
or with a normal diet, hemoglobinuria did not
occur. Our experiments involved a comparison of
the degree of hemoglobinuria induced by injection
with various levels of J or another water-soluble
vitamin K, namely 2-methyl-1,4-naphtho-quinone
sodium bisulfite (J7, or menadione sodium bisul-
fite). The specificity of vitamin E in protecting
against such hemolysis was studied. Female rats
were fed a vitamin E-deficient diet (Gyérey,
Vitamin Methods, vol. 11, 1951, p. 153) for 3
months. Half of the group were fed 1 mg of dl-
alpha-tocopherol daily. Intramuscular injection
of 75 to 175 mg/kg of J or IT caused increasing but
rather variable erythrocyte hemolysis in the defi-
cient rats but none in the tocopherol-supple-
mented animals. Colorimetric assays of the urines
for hemoglobin with benzidine dihydrochloride
showed the hemoglobinuria to be about 4 times as
severe for JJ as for an equal weight of J, or 2.5
times on an equimolar basis, calculated for anhy-
drous material in both cases. An oral dose of 20 mg
of dl-alpha-tocopherol, given to the deficient rats
4hr. prior to injection of either vitamin K deriva-
tive, protected completely from hemoglobinuria.
Feeding of 0.02% N,N ‘diphenyl-p-phenylenedi-
amine in the diet for 1 wk. gave similar protection.

1834. Pancreatic lipase, a new tool for deter-
mining the structure of triglycerides.
F. H. Marrson. Research Div. Procter and
Gamble Co., Cincinnati, Ohio.

Recent studies on pancreatic lipase have indi-
cated a specificity of this enzyme for the primary
hydroxyl groups of glycerides. Using substrates of
known composition, 2-oleoyl dipalmitin, 2-oleoyl
distearin, 2-palmitoyl diolein, and 1-oleoy] dipal-
mitin, it has been possible to establish unequiv-
ocally this specificity. Moreover, this specificity
is not influenced by the usual long chain fatty
acids as shown by studies with lard and random
rearranged lard. Under the chosen conditions
pancreatic lipase hydrolyzes only the fatty acids
esterified with primary hydroxyl groups of glyc-
erol. The use of this enzyme was thus suggested
as a tool for determining the structure of glyc-
erides. The essentials of the method consist of
a) enzymatic digestion of the triglycerides, b) iso-
lation of the digestion products, and c) characteri-
zation of the fatty acids in the isolated digestion
products. Some of the details of the method will
be described and the results obtained when the
method was applied to several natural fats will
be reported.

1835. Goldthioglucose obesity and regulation
of food intake in mice. JEAN Mayer, NoRMAN
B. MARSHALL* AND JAMES ANLIKER.* Dept. of

AMERICAN INSTITUTE OF NUTRITION

563

Nutrition, School of Public Health and Dept. of
Physiology, School of Medicine, Harvard Univ.,
Boston, Mass.

Goldthioglucose administered at the LDs5o
(1 mg/gm body wt.) causes destructive lesions
in the ventromedial nuclei of the hypothalamus
(Proc. Soc. Exper. Biol. & Med. 90: 240, 1955).
Goldthiomalate, although as toxic, does not cause
obesity nor produce lesions. To further study the
specificity of this effect, gold sodium thiosulfate
(doses of 0.5 and 1.0 mg/gm) and aurothioglyco-
anilide (doses of 0.8, 1.5 and 3.0 mg/gm) were
tried and found ineffective in producing obesity.
Sodium thioglucose (dose 2 mg/gm) protects
mice against simultaneous injections of gold-
thioglucose. In the rat, goldthieglucose does not
cause obesity, but it (like other gold compounds)
is much more toxic than in the mouse; a dose of
1 mg/gm kills all animals within 3 days. However,
if the animals are killed after 24 hr. they are found
to exhibit hypothalamic lesions involving the
periventricular and ventromedial areas. When
goldthioglucose obese and hypothalamic obese
mice are studied by an operant conditioning tech-
nique, cumulative records of food ingested do
not show the 24-hr. cycles that are characteristic
for normal mice. The rate of ingestion, however,
does not exceed the maximum rate exhibited by
normal animals. These findings are consistent with
the idea that the ventromedial area contains
‘glucoreceptors’ the destruction of which releases
the otherwise constantly activated ‘feeding
center’ from the inhibitory mechanism that
mediates the 24-hr. satiety cycles.

1836. Tea delays dental caries. J. F. McCLen-
DON AND J. GERSHON-CoHEN.* Albert Einstein
Med. Ctr., Northern Div., Philadelphia, Pa.
Although fluorine in water is cariostatic, we

knew of no proof that fluorine in ordinary foods

is; therefore, we tested tea. Ten litter-mate pairs
of Wistar rats, 21 days old, averaging 37 gm., were
one of each pair placed on diet A: 73 gm. yellow
corn, 12 sucrose, 4 corn oil, 3 liver powder, 3 lin-
seed meal, 1.5 alfalfa meal, 1 active dry yeast,

1 sodium chloride, .5 lysine, .5 tryptophan, .5

methionine and .0005 niacin. The other of each

pair was given diet A plus 20% Standards Brands
instant tea containing 170 ppm. fluorine, making

the diet 35 ppm. fluorine. After 40 days on diet A

the average was 1.5 carious teeth per rat of 92 gm.

Rats on the tea diet were free from caries and

averaged 55 gm. The fluorine content of diet A

was 1 ppm. and the rats femurs 165 ppm. The rats

femurs on the tea diet were 1700 ppm. In order to
further increase the fluorine content, tea and its
closest relative, Camellia, were planted in soil
containing 20000 ppm. fluorine. The tea plants
all died so the Camellia had to be partly substi-
tuted in the following experiment. Of 10 pairs of

564

21-day-old rats averaging 38 gm, one of each pair
was fed diet A and the other diet A plus 5% instant
tea and 5% Camellia leaves containing 2000 ppm.
fluorine. This diet contained 135 ppm. of fluorine.
After 112 days on diet A the rats averaged 102 gm
with 4.6 carious teeth. Those on the tea-Camellia
diet averaged 81 gm and 1.5 carious teeth.

1837. Dietary necrotic liver degeneration:
comparison of peripheral intravenous and
intraportal injection of vitamin E in respi-
ratory decline. WALTER MERTz aNpD K1iaus
Scuwarz (introduced by GrorcE M. BriaGs).
Nail. Insts. of Health, Bethesda, Md.
Respiratory decline, a metabolic defect in

normal-appearing liver slices, preceding acute

necrosis in rats on vitamin E-free Torula yeast

diets is not influenced by addition of vitamin E

to the Warburg medium. However, it is reversed

by intraportal injection of tocopherol emulsions

(RopnNAN, et al Federation Proc. 14: 270, 1955, and

J. Biol. Chem. In press). The reversion of the

defect is measured by comparing the rate of

respiratory decline of post-injection slices to that
of preinjection slices. The latter are prepared
from a liver lobe extirpated under ether anes-
thesia immediately before the injection. Inbred

(Fisher 344) rats were used after 15-17 days on

the basal diet. In this strain, intraportal injection

of 1507 of emulsified DL-a-tocopherol at the

time of operation was found to produce a 47%

reversion of the decline (av. of 10 animals) after

30 min. Injection of various doses of the tocopherol

emulsion into the jugular vein gave the following

results: 200 7: 94% (10 animals), 150 7: 55% (9),

100 y: 35% (10), and 0: 0% (2) reversion. Thus,

the peripheral injection of tocopherol emulsions

leads to effects not significantly different from
those obtained by injection into the portal vein.

1838. Alleviation of molybdenum toxicity
in the rat with inorganic sulfate. RussELL
F. Mriiter anp NExson O. Price (introduced
by Paut H. Puruurps). Dept. of Biochemistry
and Nutrition, Virginia Polytechnic Inst.,
Blacksburg.

The addition of 75 or 100 parts per million
(ppm) of molybdenum as H2MoOQ, or Na2MoO, to
the basal ration, which contained no inorganic
sulfate, caused a growth depression in the rat.
This toxic action of molybdenum could be al-
leviated by the inclusion in the diet of 2,200 ppm
of sulfate (equimolar mixture of NaSO, and
K.SO,). The composition of the basal ration fol-
lows: 80.5% sucrose, 12% casein, 5% cottonseed
oil, 2.5% nonsulfate salts, and 0.2% .w-cystine.
Vitamins to meet the requirement of the rat were
fed. To this basal ration was added 3.2 ppm of
copper so that the ration averaged 4 ppm in
copper. The sulfate supplement added less than

FEDERATION PROCEEDINGS

Volume 1§

0.2 ppm of copper. Blood and liver copper and
molybdenum concentrations were increased when
the molybdenum supplement was fed, without
added sulfate. Lesser concentrations (particularly
copper) were observed when the molybdenum-
containing diet was supplemented with sulfate.

1839. Effect of hydrocortisone on ascorbic
acid synthesis in the rat. AGNES Fay Moraan
AND Miuprep J. Brennert.* Dept. of Home
Economics, Univ. of California, Berkeley.
To test the hypothesis that the glucocorticoid

fraction of the adrenal hormonal output plays a

role in the synthesis of ascorbic acid in the rat, a

comparison of the urinary and tissue ascorbi¢

acid of normal and adrenalectomized rats was
undertaken. Hydrocortisone was administered to
both normal and adrenalectomized rats during
the 2 wk. before killing, which was 4 wk. after
adrenalectomy. Blood glucose, liver glycogen,
adrenal and thymus weights were used as indices
of hydrocortisone action. Removal of the adrenal
glands resulted in decreased urinary and liver
ascorbic acid. The vitamin C levels of the other
tissues did not change. Daily administration of
hydrocortisone for 2 wk., starting 4 wk. after
the operation, caused the urinary and _ liver
ascorbic acid levels to return almost to normal.
This indicates that the hormone at least facilitates
the synthesis of the vitamin. It does not appear to
be obligatory, inasmuch as the other tissues were
not affected and adrenalectomized rats maintained
on a vitamin C-free ration for 220 days showed
no scorbutic symptoms. It is postulated that the
mechanism whereby the hormone facilitates the
synthesis of ascorbic acid is either a) through the
carbohydrate supply (liver glycogen) or b) by
blocking glucose oxidation and thus accelerating
its conversion to ascorbic acid. The interrelation-

ships of ascorbic acid, glutathione, coenzyme A,

ACTH, and the adrenal glucocorticoids are

considered.

1840. Ash of unidentified growth factor sup-
plements. A. B. Morrison,* RicHarp Dam,*
L. C. Norris anp M. L. Scorr. Cornell Univ.,
Ithaca, N.Y.

Previous experiments (Pouliry Sci. 34: 738,
1955) showed that the ash of a mixture of un-
identified growth factor supplements, consisting
of distillers dried solubles, fish solubles, forage
juice, dried whey product and penicillin mycelium
meal, or of distillers dried solubles, significantly
increased chick growth at 4 wk. of age when fed
in a purified diet. The diet contained adequate
quantities of all nutrients known to be required.
The inclusion in the diet of addition amounts of
the known essential minerals failed to stimulate
growth. Further studies using chicks of depleted
hens have confirmed the original observations,

ces
ex
th:
alf
the
the
oat
inc

tio
siu

int
tha
hot
tio
fro)

ume 1§

er and
1 when
vithout
cularly
lenum-
sulfate.

corbic
[ORGAN
| Home
ley.

rticoid
plays a
e rat, a
scorbic¢
‘ts was
ered to
during
<. after
ycogen,
indices
adrenal
d liver
e other
tion of
. after
1 liver
normal,
ilitates
pear to
es were
ntained
showed
hat the
tes the
ugh the
- b) by
lerating
elation-
yme A,
ids are

yr sup
. Dam,*
l Univ.,

4: 738,

of un-
nsisting
, forage
ycelium
ficantly
hen fed
dequate
equired,
punts of
imulate
lepleted
vations,

March 1956

and indicate that the mineral(s) involved are
concerned in bone formation. Highly depleted
chicks fed the basal diet exhibited leg-bone mal-
formation characterized chiefly by enlargement
of the hock joint. The ash of the mixture of un-
identified factor supplements markedly reduced
the incidence of the syndrome. The percentage ash
in the tibiae of chicks fed the basal diet was sig-
nificantly (P < 0.02) lower than that of chicks of
equal weight fed the diet plus ash (45.07% and
47.30%, respectively). No effect of treatment on
tibia length was observed. Thirty-two nonessen-
tial elements, fed alone or in combination, failed
to increase growth. Feeding a mixture of reagent
grade minerals in the amounts indicated by
spectrographic analysis of the ash did not pro-
mote growth, but Hoagland’s A-Z mixture was
partially effective. Bacteriological studies re-
vealed no consistent effects of the ash on the
intestinal microflora.

1841. Factors controlling excretory pattern
of potassium (K-42) and cesium (Cs-134)
in rats. F. R. Mraz* anp H. Parricx. Univ.
of Tennessee-Atomic Energy Commission Agric.
Research Program, Oak Ridge, Tenn.

A difference in excretory pattern was observed
in studies dealing with the influence of potassium
chloride, cortisone, parathyroid extract and di-
enestrol diacetate on metabolism of cesium. When
a purified diet was fed to rats, approximately
25 times more cesium-134 was excreted in urine
than in feces, however, when a diet containing
natural feedstuffs was fed, urinary excretion of
cesium-134 was only 2-6 times greater than fecal
excretion. Fractionation studies demonstrated
that a factor(s) found in oat hulls, wheat bran,
alfalfa meal and crude soybean oil meal reduced
the excretion of cesium and potassium by way of
the kidneys, whereas corn, corn starch, rolled
oats, wheat flour or purified soybean protein
increased the urinary excretion of cesium-134
and potassium-42 and reduced their fecal excre-
tion. The total excretion of cesium-134 or potas-
sium-42 remained relatively constant.

1842. Effect of aureomycin on apparent util-
ization of vitamin A by rat. T. K. Murnay*
AND J. A. CAMPBELL. Food and Drug Labs., Dept.
of Natl. Health and Welfare, Ottawa, Canada.

In a previous paper (J. Nutrition 57: 89, 1955)
it was postulated that, while aureomycin increased
the response of rats to vitamin A, as measured by
the vaginal smear assay, it did not do so by in-
treasing absorption of the vitamin from the
intestinal tract. This was based on the observation
that aureomycin was effective even when it was
not fed until 24 hr. after dosing and on the assump-
tion that all vitamin A would have disappeared
from the gut contents within 24 hr. after an oral

AMERICAN INSTITUTE OF NUTRITION

565

dose. This assumption has now been shown to be
a fact. Furthermore, aureomycin, given orally,
increased the response to subcutaneous doses of
vitamin A, although no part of these doses ap-
peared in the intestinal contents or wall. Aureo-
mycin increased the response to doses of vitamin
A when the vitamin A free diet was supplemented
with a vitamin mixture but was ineffective when
the diet was supplemented with ascorbic acid,
folic acid or when sucrose was substituted for
starch. The implications of these observations
will be discussed.

1843. Comparison of nitrogen balance on
amino acids and whole egg. E. 8. Nassgt.
Dept. of Physiology, Univ. of Rochester School
of Medicine and Dentistry, Rochester, N. Y.
Nitrogen balances were determined on adult

male rats fed various diets by stomach tube.

Isonitrogenous and isocaloric diets containing

either crystalline amino acids or whole egg were

compared. Each diet was tested in a feeding cycle

of 5 wk-2 wk. on maintenance diet; 1 wk. on a

nitrogen-free diet; 1 wk. of experimental diet

containing approximately half of the maintenance
nitrogen requirement; 1 wk. on twice the nitrogen
intake of the previous week. The ‘complete’ amino
acid mixture simulated whole egg in the essen-
tials. The ‘new’ mixture was based on the essen-
tial amino acid requirements, determined singly
in previous work. The nitrogen balance index of
ingested nitrogen, K’, is 1.27, 1.08, 1.24 for ‘com-
plete’ AA mixture and 2 whole egg experiments
respectively; 0.59 and 0.64 for 2 experiments with
the ‘new’ AA mixture. The ‘new’ mixture, which
conceivably might have been utilized with highest
efficiency, is in fact less well utilized than the
‘complete’ mixture which simulates whole egg.

1844. Effect of a 36-hr. period of pteroyl-
glutamic acid (PGA) deficiency on fetal
development in the rat. Marsorie M.
Neutson, Howarp V. Wricut,* CATHERINE
D. C. Barrp* anp HERBERT M. Evans.* Inst.
of Exptl. Biology, Univ. of California, Berkeley.
Previous studies have shown that multiple

congenital abnormalities or fetal death can be

produced in the rat in high incidence by a transi-
tory PGA-deficiency period of only 48 or 72 hr.,
but not 24 hr., during the early part of pregnancy.

The earlier phases of embryonic development were

more severely affected by the same length of

deficiency than were the later phases. In order to
determine more accurately the length of time
required for PGA-deficiency to affect fetal de-
velopment and to find the period of greatest
sensitivity, the effects of a 36-hr. period of de-
ficiency instituted on the 7th and 8th days of
gestation were determined and compared with
those for the corresponding 24-hr. periods. When

566

the PGA-deficient diet containing 1% SST and
0.5% x-methyl PGA was given for 24 hr., begin-
ning on either the 7th or 8th days, only 10-11%
of the implantation sites showed abnormal fetal
development or fetal death. In contrast, the 36-hr.
period of deficiency markedly affected fetal
development. When the PGA-deficient diet was
instituted on the 8th day, 80% of the embryos
were injured, whereas only 33% were affected
when the diet was started on the 7th day. The
difference in sensitivity of the earlier embryonic
tissue to PGA-deficiency may be explained by a
decreased susceptibility of the undifferentiated
cells or by their greater regulatory power.

1845. Effect of acidosis on intracellular com-
position in normal and diabetic rats.
Nancy NIcHOLS AND GEORGE NICHOLS, JR.
(introduced by Howarp F. Root). Baker Clinic
Research Lab. and Dept. of Medicine, Harvard
Med. School, Boston, Mass.

The effect of acidosis on intracellular glycogen
and electrolytes was determined in livers and
muscles of non-diabetic and diabetic rats fasted
20 hr. Effects of glucose feeding on acidosis were
also observed. No ketosis was present in fasted
or fed animals. In most instances, acidosis caused
inverse changes in liver and muscle. Diabetics
had higher liver (13.2 gm/kg ICW) and muscle
(1.47) glycogen than non-diabetic liver (1.17)
and muscle (1.31). Acidosis increased glycogen
content in non-diabetic livers (2.67) and decreased
it in diabetic livers (10.5), non-diabietic muscles
(1.08) and diabetic muscles (1.14). Glucose in-
creased liver and muscle glycogen in normals and
decreased it in diabetics. Acidosis in fed rats
caused decreased liver glycogen in non-diabetics,
and decreased liver and muscle glycogen to almost
zero in diabetics. Glycogen increased in non-
diabetic, fed, acidotic muscles. In fasted non-
diabetics, liver potassium fell (170-162) in aci-
dosis, while muscle potassium rose (165-172).
Diabetics had high liver potassiums (179) and
low muscle potassium (159). Acidosis did not
affect liver potassium (178) but lowered muscle
potassiym further (150). Liver phosphate was low
in diabetic acidotics and increased in non-
diabetic and diabetic muscle. Acidosis increased
calcium and magnesium levels in non-diabetic and
diabetic muscles and in non-diabetic livers. They
decreased in diabetic acidotic livers. All of the
electrolyte abnormalities caused by acidosis were
minimized c” abolished by glucose administration,
in both non-diabetics and diabetics, despite the
fact that there was no evidence for glucose utiliza-
tion in the diabetic rats. (Supported by PHS
Grant A-520.)

1846. Effect of tocopherol on creatinuria of
children with steatorrhea. H. M. Nitowsky,*

FEDERATION PROCEEDINGS

Volume i

H. H. Gorpon anv J. T. Trnpon.* Sinai Hosp,

of Baltimore, Harriet Lane Home and Dept. of

Pediatrics, Johns Hopkins Univ., Baltimore, Md

Studies of erythrocyte hemolysis in hydrogen
peroxide and of plasma tocopherol of childrep
with cystic fibrosis of the pancreas and biliary
atresia indicate the existence of a prolonged
deficiency of vitamin E. Since increased creati-
nuria has been noted before muscular weakness in
E deficient rabbits (MacKEnz1g anD McCo.uuw),
studies were made of the effect of tocopherol ad-
ministration on creatine excretion of 5 children,
age 3-103 yr., with cystic fibrosis of the pancreas,
and of 2 infants, age 5 and 9 months, with biliary
atresia, while on creatine-poor diets. The adminis.
tration of relatively large doses of tocopherol
esters raised plasma tocopherol from 0-0.2 to
0.6-2.8 mg%. Erythrocyte hemolysis fell from
54-100% to 0-2%. In 3 children with fibrocystic
disease, urinary creatine decreased from 5.1, 86
and 8.8 to 0.8, 1.7 and 2.4 mg/kg/day, with no
significant alteration in creatinine excretion,
Creatine output, expressed as percentage of total
creatinine for these subjects, and for two others
for whom complete 24-hr. urines were not ob-
tained fell from 24-40% to 7-15%. In two infants
with biliary atresia, reversal of hemolysis and 4
marked rise of plasma tocopherol were obtained
only after intravenous administration of tocoph-
erol polyethylene glycol-1000-succinate; no altera-
tion of creatine excretion was noted. Further
studies are being made of the mechanisms by
which tocopherol decreases creatinuria in patients
with cystic fibrosis of the pancreas.

1847. Vitamin By. deficiency and fasting on
distribution of P® in new-born rats. B. L,
O’De.L, J. H. BRUEMMER* AND A. G. Hogan,
Dept. of Agric. Chemistry, Univ. of Missouri,
Columbia.

New-born rats from vitamin By-deficient dams
and new-born from B,:-supplemented dams, some
of which were fasted from birth, were injected
with 10 we of P%?, After 12 hr. the brains and livers
were fractionated according to the method of
Schmidt and Thannhauser. The radioactivity
and phosphorus were determined and the results
expressed as specific activity (SA) and relative
specific activity (RSA). RSA was related to the
acid soluble fraction. The SA of the acid soluble
fraction of liver was higher in both the By-de-
ficient and the fasted animals. The vitamin de-
ficiency had no effect on the RSA of the lipid
fractions, but fasting lowered it in both tissues
to 30% of the litter-mate controls. The RSA of
the nucleic acid fractions (DNA and PNA) of
both liver and brain were significantly lower
(20-30%) in the deficient animals. Thus the rate
of phosphorus turnover in DNA was lowered by

— Ss S&S SDR wD DD west ee

= Ss om st S$ oO oso @

“olume tj

vat Hosp,
Dept. o
nore, Md,
ry drogen
childrep
1 biliary
rolonged
d creati-
aukness in
SOLLUM),
herol ad-
children,
yancreas,
h biliary
adminis.
copherol
0-0.2 to
ell from
orocystie
1 5.1, 86
with no
xcretion,
- of total
‘oO others
not ob-
> infants
is anda
obtained
_tocoph-
o altera-
Further
isms by
patients

ting on
s. B.L,
Hogan,
Tissouri,

nt dams
ns, some
injected
nd livers
thod of
activity
e results
relative
d to the
| soluble
} Byo-de-
min de-
he lipid
1 tissues
RSA of
-NA) of
y lower
the rate
vered by

March 1956

the vitamin By» deficiency although the corcen-
tration of DNA per nucleus was unchanged.
Fasting had a similar but more marked effect on
phosphorus turnover in the nucleic acid fractions.
The RSA of the liver PNA was reduced to 25%,
and of the DNA to 50% of the controls. The corre-
sponding values for brain were 50% each. While
fasting affected nucleic acid metabolism in the
same manner as a By deficiency, there was a
marked difference in its effect on phospholipid
metabolism. (Supported in part by grant no.
A 352 from the Natl. Insts. of Health.)

1848. Bone density relationships in adoles-
cence. Lura M. Op.anp (introduced by AGNES
Fay Moraan). Dept. of Home Economics Re-
search, Montana Agric. Exper. Station, Bozeman.
Bone density measurements of a cross-section of

the os calcis for students 14-17 years of age aver-

aged 0.80+0.01 for 98 boys and 0.64+0.01 for 87

girls. For these subjects density values for the end

section of the phalanx 5-2 were 1.11+0.03 for the
boys and 1.18+0.03 for the girls. Center phalanx
cross-sectional values were somewhat higher;
1.63+0.04 for boys and 1.83+0.04 for girls. These
values are reported as number of x-ray equivalent
gm bone ash/cec bone. There were significant sex

differences for both os calcis and phalanx 5-2

measurements. There were significant positive

relationships between bone density and chrono-
logical age for these subjects. There was a positive
relationship between bone density values of the

end and cross-sectional values of the phalanx 5-2,

but there was little evidence of a strong relation-

ship between os calcis and phalanx 5-2 bone
density values. No relationship was evident be-
tween bone desnity and the intake of certain
nutrients as calculated from current 7-day dietary
records. Significant relationships were observed
for certain indices of physiological age. (Cooperat-
ing agencies: Human Nutrition Research Branch,
USDA and the College of Chemistry and Physics,
Pennsylvania State Univ.)

1849. Food intake and sex hormone effects
on serum and liver cholesterol. RutH OKEY
AND Martan M. Lyman.* Dept. of Home Eco-
nomics, Univ. of California, Berkeley.
Increasing the protein content of a cholesterol-

tich diet from 15% to 30% resulted in a greater de-

crease in liver cholesterol storage in male than in
female rats. In order to test whether this differ-
ence was due to a specific hormone effect or re-
flected only the greater food intakes and growth
tates of the males, the following studies were
made. Three groups of ten 150-gm male rats were

placed on the 15% protein diet and 3 on the 30%.

Two groups on each diet were castrated and one

castrate group on each diet was given 0.05 mg

estradiol benzoate 3 times weekly. At autopsy

eaa6 the

AMERICAN INSTITUTE OF NUTRITION

567

after 3 wk. it was found that castration had de-
creased the differences in liver cholesterol associ-
ated with the protein content of the diet, and the
hormone dosage had decreased them still further.
However, the hormone depressed growth rate and,
to a lesser extent, food intake. Therefore the ex-
periment was repeated with a hormone dosage 3
lower. Additional control and castrate groups whose
food intake was restricted to that of the hormone-
dosed rats were included. The ad libitum fed rats
showed liver cholesterol differences of the same
type as those previously observed. Restriction of
food intake, as well as hormone dosage, resulted in
an increase in serum cholesterol. This was most
marked in the control and castrate groups fed 30%
protein. Simultaneously, liver cholesterols de-
creased in the restricted animals untreated with
hormone. (Supported in part by PHS grant-in-aid
H-1013.)

1850. Absorption of vitamin B,. in hyper-
and hypothyroid rat. Kunio Oxupa,* San-
FORD STEELMAN* AND Bacon F. Cuow. Dept.
of Biochemistry, School of Hygiene and Public
Health, Johns Hopkins Univ., Baltimore, Md. and
Armour Research Lab., Chicago, Ill.
‘Absorption of orally administered radioactive

B,2 was studied in rats in different states of thyroid
activity. Fifty mug of Co®-labeled Biz were given
to test animals by stomach tube, and feces were
collected for 6 days for the determination of
radioactivity. The difference between the adminis-
tered and excreted radioactivity is taken as a
measure of absorbed Biz. Our data show that
thyroidectomy resulted in a decrease of absorption
from 25.5+1.0 wg (before thyroidectomy) to
6.2+1.1 ug (after thyroidectomy). When the thy-
roidectomized rats were subsequently given a
stock diet containing 0.1% desiccated thyroid for
3 wk., the absorption was increased to normal
levels (27.741.3 ug). To study the effect of thyroid
treatment on rats with normal thyroid activity,
stock animals were given a casein-containing diet
supplemented with 0.2% desiccated thyroid for 5
wk., after which the absorption test was again
carried out. It was found that such treatment de-
creased fecal excretion from 26.0+1.0 ug for the
controls to 18.3+1.9 ug for the treated animals.
This increase in absorption was also reflected in
the greater content of radioactivity in various
organs, being 7.00.2 myg vs. 3.4+0.4 mug in liver
and 4.7+0.3 mug vs. 3.2+0.4 mug kidney. The
larger values were in each case for thyroid treated
rats.

1851. Absorption of basic amino acids from
human small intestine. ALINE UNDERHILL
OrtEN, Nicuotas S. GimBeL* anp ArTHUR H.
Smitu. Depts. of Physiological Chemistry and

568

Surgery, Wayne Univ. College of Medicine, De-

troit, Mich.

Two human subjects are being studied with
respect to the absorption of amino acids from
permanent Thiry loops of small intestine. The
present report deals with the absorption of the
basic amino acids. The amino acid analyses are
being carried out by column chromatographic
techniques. When a commercial protein hy-
drolysate was employed, it was found that the
percentage of absorption of the basic amino acids
in decreasing order was arginine, lysine or trypto-
phane, then histidine. When a similar study was
made of an equimolar mixture of 17 pure L-amino
acids, the same pattern of absorption for the basic
amino acids was obtained. However, when an
equimolar mixture composed of the 4 basic amino
acids alone was tested, the absorption of lysine
was markedly reduced. Various combinations of
the individual basic amino acids are being investi-
gated. These results together with those previ-
ously obtained with leucine, isoleucine, and
phenylalanine will be discussed. (Supported by
grant A-693 from the Natl. Insts. of Health and by
a grant from the Detroit Receiving Hosp. Re-
search Corp.)

1852. Factors in liver reversing thyroid stress
in rats. Atwoop C. PaGgE, JR.,* FRANK R.
Konruszy,* Donaup E. Wotr,* Paut ALDRICH*
AND Karu Foukers. Research Labs., Chemical
Div., Merck & Co., Rahway, N. J.

The fractionation of crude liver preparations
was undertaken in an effort to isolate the factors
responsible for the antithyrotoxicosis activity ob-
served originally by Ershoff in weanling rats. The
solubilization of the substances having the full ac-
tivity of liver insolubles was very difficult. How-
ever, components giving an appreciable biological
response were extractable with pyridine or am-
moniacal ethanol solution. The first active concen-
trates which were fractionated from such prepa-
rations contained predominately unsaturated
fatty acids. It was demonstrated that other
sources of unsaturated fatty acids were equally
effective; thus, unsaturated fatty acids were
identified as one of the components in liver having
activity. Other concentrates, the activity of which
was not attributable to unsaturated fatty acid
content, were fractionated and found to contain
appreciable amounts of cholesterol. Cholesterol
and a variety of steroids including the bile acids
were found to be active; thus, a steroid component
also increases growth in this thyroid stress assay.
The effects of these two groups of known com-
pounds, unsaturated fats and steroids, in the rat
test were essentially additive and accounted for an
appreciable fraction of the response observed
with liver supplements. Nutritional studies of

FEDERATION PROCEEDINGS

Volume ti8 Ma

liver supplementation under isonitrogenous con-Btijo
ditions led to the observation that added caseinf ths
or soybean protein as well as liver could improyh (a
growth. The combination of corn oil, bile acids andfi ¢ |
casein was not quite as effective as liver insolubles§ pia
indicating the presence in crude source materiakg |i
of another substance promoting growth in this
thyroid stress assay.

1853. Absorption of fructose from intestinal
tract of rat. NicHoLaAs M. PAPADOPOULOS* ANDE,
JosePpH H. Ror. Biochemistry Dept., School of.
Medicine, George Washington Univ., Washington,
D.C.

Homogenates of intestinal mucosa of normal
rats in phosphate buffer, po 7.8, mixed with ATP
and fructose and incubated at 30° showed the for.
mation of fructose-6-phosphate and fructose-1,6
diphosphate. Incubation of the same mixture, with
fructose replaced by fructose-1,6-diphosphate and
ATP omitted, yielded fructose-6-phosphate and
fructose. Phosphate esters were identified by paper
chromatography and by copper reduction before
and after 7 min. hydrolysis at 100° with n HC.
Analysis of trichloroacetic acid extracts of in-
testinal mucosa removed from rats after Nembutal
anaesthesia and intraduodenal injection of frue-
tose solution showed the presence of the same
fructose esters. Balance studies indicate that most
of the fructose in the intestinal lumen reappears in
the portal blood as free fructose; some loss of free
fructose occurs, possibly by glycolysis, or trans-
formation into glucose, or transfer into a non
glycolytic pathway. The methods used showed the
presence of small amounts of fructose phosphate
esters, but not free fructose, in the intestinal
mucosa and blood of the fasting rat. The amounts
of fructose esters in the mucosa are increased
markedly during fructose absorption. Since both
blood and mucosa have dephosphorylating ac-
tivity, there is very little increase in fructose
phosphate esters, but considerable accumulation
of free fructose, in portal blood, during fructose
absorption. The data obtained show by dirett
analysis that phosphorylation of fructose and de
phosphorylation of fructose phosphate esters occur dee
in the intestinal mucosa during fructose ab fie
sorption. Ing

blo
imk
pla:

1854. Supplemental value of raw, lime treated
and boiled corn when added to a niacin-})t
free low casein diet. Wixu1aM N. Pearson,} id
J. SatvaporR VALENZUELA AND Saran Janey (og
Sremprex (introduced by W1L.1aM J. Darsy).§\P
Div. of Nutrition, Depts. of Biochemistry anijtior
Medicine, Vanderbilt Univ. School of Medicinejiat
Nashville, Tenn. N¢
When fed as a supplement to diets low in casein} !.lé

and niacin, ‘limed’ corn (tortilla) is reported to be}!00

superior to raw corn for rat growth. Supplementa-|The

mal

Volume ij

nous Cop.

nsolubles,
materiak
h in this

ntestinal
JLOS* AND
School of
ashington,

f normal
with ATP
d the for
ctose-1,6
ture, with
phate and
hate and
| by paper
on befor
h n HCL.
ts of in
Nembutal
1 of frue-
the same
that most
\ppears in
ss of free
or trans-
Oo & NOD
owed the
yhosphate
intestinal
- amounts
increased
ince both
ating ac:
fructose
mulation
+ fructose
by direct
e and de
ters occur
‘tose ab-

p treated
- niacin:
PEARSON,
AH JANE
Dakgsy),
istry and
M edicine,

in caseiD
rted to be
rlementa-

March 1956

tion of raw corn with niacin increases growth to
that obtained with limed corn (LAGUNA AND
CARPENTER, J. Nutrition 45: 21, 1951; Cravioto
eal. J. Nutrition 48: 453, 1952). Liberation of
niacin from a bound form during preparation of
limed corn has been suggested as an explanation
of this effect (KopiceK, Biochem. J. 48: viii, 1951).
In the present study weanling female Sprague-
Dawley rats were fed a niacin-deficient, 9% casein
diet for 30 days. Raw, limed, or boiled corn was
incorporated at a level of 40% of the diet (replac-
ing an equivalent amount of sucrose). Both limed
and boiled corn permitted better growth than did
raw corn. Supplementation with niacin (8 mg %)
or DL tryptophane (400 mg %) increased growth
substantially when the diet contained raw corn,
but not when it included limed or boiled corn.
Microbiological assay of limed and boiled corn re-
vealed niacin losses up to 40% during processing
but no significant decrease in tryptophane content.
Since boiling alone is sufficient to increase the nu-
tritional value of corn it would appear that the
role of lime is unimportant. (Supported in part by
agrant from E. I. du Pont de Nemours and Co.)

1855. The amino acid inadequacy of blood.
H. S. Perpus,* G. F. Lamspert* anp D. V.
Frost. Abbott Labs., North Chicago, Ill.
These studies further evaluate human blood and

plasma and canine blood as sources of essential

amino acids for the protein depleted adult male

rat (Proc. Soc. Exper. Biol. & Med. 86: 742, 1954).

All three failed to support satisfactory repletion

rates when rats were offered 120 mg N per day with

non-protein diet. The inadequacy of whole blood
proved much more profound than that of human
plasma. Addition of L-isoleucine and pui-methi-
onine accelerated repletion dramatically. Sepa-
rate additions had less effect. Human blood sup-
ported good gains when supplemented with 55 mg
Lisoleucine and 15 mg pu-methionine/120 mg of
blood N. The question comes whether amino acid
imbalance contributes to the incompletely ex-
plained azotemia accompanying gastrointestinal
bleeding in humans. Feeding dogs hemoglobin pro-
duced a greater increase in blood urea N than

feeding casein and beef (Surgery 10: 991, 1941).

Ingestion of 800 ml of blood produced azotemia in

man (Am. J. Med. Sct. 211: 565, 1946). In our ex-

periments, feeding normal dogs a non-protein diet
and 150-375 mg N/kg/day as lyophilized human or
dog blood did not increase the blood urea N or

NPN or produce negative N balance. Administra-

tion of 12-25 ml of citrated human blood by gastric

intubation produced an increase in the blood urea

N of fasting rats. Supplementing the blood with
115 gm t-isoleucine and 0.31 gm pi-methionine/

100 ml, however, did not reduce the blood urea N.

The possible relation of amino acid imbalance to

AMERICAN INSTITUTE OF NUTRITION

569

azotemia in gastrointestinal bleeding requires
further clarification.

1856. Depression of plasma cholesterol in
human subjects consuming butter contain-
ing soy sterols. D. W. Peterson, C. W.
NicHo.s, Jr., N. F. Peek anp I. L. CHarkorr
(introduced by C. R. Grav). College of Agricul-
ture and School of Medicine, Univ. of California,
Davis and Berkeley.

Experimental subjects were allowed to consume
their normal three meals a day with the addition,
during the control period, of 14 gm of ordinary
butter (OB), or, during the experimental period, of
14 gm/meal of butter containing 1.9 gm of soy
sterols (SSB), a total of 5.7 gm of soy sterols/day.
Blood samples were drawn and analyzed for total
plasma cholesterol once a week during each period
and 3 times a week at 2-day intervals during the
terminal week of any period. These samples were
taken 1-1} hr. after breakfast. Group A, period I:
Ten subjects consumed OB for 1 wk. Mean plasma
cholesterol (3 samplings) was 218 mg %; period
IT: the subjects consumed SSB for 3 wk. Mean
plasma cholesterol (3 terminal samplings) had
dropped to 194 mg %; period III: subjects were
returned to OB. During the 3rd week (3 ter-
minal samplings) plasma cholesterol had risen to
209 mg %. Group B, period I: Nine additional
subjects were fed OB for 4 wk. Mean plasma
cholesterol (3 terminal samplings) was 217 mg
%; period II: subjects consumed SSB for 3 wk.
Mean plasma cholesterol (3 terminal samplings)
had dropped to 194 mg %. In both groups differ-
ences of 11% or greater were found. The differences
were significant at the 1% level. (Supported in
part by the American Heart Assn.)

1857. Relation of fluorine to metabolism of
sulfur-35 and related factors. J. J. Prna-
man,* J. A. Bacon,* F. R. Mraz* anv H. Pat-
rick. Univ. of Tennessee-Atomic Energy Com-
mission Agric. Research Program, Oak Ridge.
Studies dealing with the metabolism of inorganic

sulfur were conducted with the rat and the chick.

Rats that were depleted in vitamin By: and folic

acid during gestation and lactation, as well as

rats weaned from mothers on a nutritionally ade-
quate diet, were utilized in the rat studies. The
influence of different experimental diets deficient
in methionine or factors associated nutritionally
with methionine on uptake of sulfur-35 by im-
planted bone, as well as normal bone and cartilage,
was observed in the chick studies. The relation of
vitamin Biz, choline, methionine, cystine, homo-
cystine, cysteine, hydroxy methionine and aureo-
mycin to uptake of inorganic sulfur-35 and also the
influence of fluorine on uptake of sulfur-35 as re-
lated to these factors, including growth response
were obtained with both chicks and rats. The

570

sulfur-containing amino acids reduced the reten-
tion of inorganic sulfur-35 in both rats and chicks.
The growth response and sulfur-35 retention in
chicks produced by vitamin Biz or aureomycin
supplementation was reduced by sodium fluoride.
Methionine and cystine gave partial protection
from sodium fluoride.

1858. Level and type of dietary fat and experi-
mental hypercholesteremia in the cebus
monkey. O. W. Portman,* F. J. STARE AND
D. Bruno.* Dept. of Nutrition, Harvard School
of Public Health, Bosion, Mass.
Hypercholesteremia, hyperbetalipoproteinemia

and atherosclerosis were produced in the cebus

monkey by feeding a purified diet containing cho-
lesterol, deficient in sulfur amino acids, and in
which 33% of the calories was provided by corn oil
and cod liver oil (Mann et al., 1953). In the present
study the serum lipid responses were studied when
different levels and types of fat were fed. These
regimens supplied equal quantities of calories,
protein and cholesterol. One group of monkeys re-
ceived 45% and the other 10% of the calories as
corn oil. The animals were maintained on these
diets for 3 consecutive 8-wk. periods. The monkeys
fed high fat diets had mean serum cholesterol
values of 370 mg % at 8 wk. compared to 251 mg %
for monkeys fed low fat diets. The effect of chang-
ing from low to high fat diets was an increase,
while the effect of changing from high to low fat
diets was a decrease in cholesteremia. Nine
monkeys fed different levels of fat as corn oil were

changed to diets containing the same level of a

hydrogenated vegetable oil. An elevation over

prevous serum cholesterol levels resulted.

1859. Pantothenic acid-sparing action of
dietary ascorbic acid in antibody produc-
tion. J. PruzaNsky* aNp A. E. AXELROD.
Biochemistry Dept., Univ. of Pittsburgh School
of Medicine, Pittsburgh, Pa.

Dietary ascorbic acid at the 2% level has been
shown to spare the requirement for pantothenic
acid in growth and survival (Darr AND ScHWARZ,
Federation Proc. 11: 200, 1952) and reproductive
performance (Everson et al. J. Nutrition 54: 305,
1954) of the rat. In this laboratory a similar
sparing action was noted in the ability of the rat
or produce antibody to diphtheria toxoid. Animals
receiving 2% ascorbic acid added to a pantothenic
acid-deficient diet were superior in growth and
antibody response to a similar group of deficient
animals unsupplemented with ascorbic acid. Not
every animal responded to the dietary ascorbic
acid. A definite correlation existed, however, be-
tween growth and antibody response. Animals
whose growth response was marked also demon-
strated significant antibody titers. The individual
data including liver, spleen and adrenal coenzyme

FEDERATION PROCEEDINGS

Volwme |

A contents of some of these animals will be py
sented.

1860. Need for pantothenic acid and its rek
tion to ascorbic acid in nutrition of guine
pig. CEecELIA PUDELKEWIcz* AND CHARLOT
RopEeruck. Home Economics Research, Tou
State College, Ames.

Nine weanling male guinea pigs fed a compleif
natural ration containing 0.15% omega-methy|
pathothenic acid for 15 days ate 1 gm more f
but gained 0.9 gm less weight each day than thei
control litter-mates. Concentrations of blooj
serum ascorbic acid of 3 experimental pigs aver!
aged 4 that of 3 controls. When the analogue wy
increased to 0.30% for 18 days, followed by 0.40%
for 14 days, differences in food intake, weight gain
and food efficiency between the two groups in
creased. Experimental animals were anemic, their
serum ascorbic acid levels averaged 34 that of
controls, and pyruvic acid accumulated in thei
blood. Symptoms included soft woolly fur, pallor,
lassitude, salivation, watering of the eyes, museu-
lar weakness of the hind legs, convulsions and
coma. Fatty livers and kidneys, hemorrhagic
adrenals and splenomegaly were observed upon
autopsy. The pantothenic acid deficiency appeared
to be chronic rather than acute. When 2- to 4-day-
old male guinea pigs were fed a complete seni-
synthetic ration along with 4 levels of ascorbic acid
(0, 2, 10, and 40 mg/day) and 4 levels of calcium
pantothenate (0, 0.06, 0.2, and 8 or 1 mg/day), ?
animals developed acute pantothenic acid de-
ficiencies. Inanition probably complicated symp-
toms suggestive of deficiency in 2 other pigs. Soft
woolly fur, lassitude, diarrhea, convulsions and
hemorrhagic adrenals occurred, but there was no
anemia and no decrease in blood plasma ascorbic
acid concentrations. No clear-cut interrelation-
ships between pantothenic acid and ascorbic acid
were observed.

1861. Protein and amino acid studies with the
guinea pig. Mary Evizasetu Rem. Natl. Insts.
of Health, Bethesda, Md.
Investigations have been conducted with guinea

pigs (starting age 2-7 days) placed on purified

diets (J. Nutrition 51: 341, 1953) containing differ-
ent levels of casein or soybean protein (Drackett)
and a mixture of carbohydrates. An average weight
of 340 gm at the end of 6 wk. was obtained witha
30% level of either type of protein. With a diet
containing 20% casein the average weight was
only 240 gm. Addition of 1% arginine resulted ina
maximal growth response. To produce the maxi-
mal response with 20% Drackett protein, addition
of a small amount of methionine was necessary.

Additional arginine was not required. Present

results indicate that the guinea pig, as compared

with the rat, has a high arginine requirement. In

pre
res

tai
ret
av
to

ee ee ee ee ee |

Volwme |

vill be pr

d its rely
of guine,
YHARLOT
rch, Tow

4 complet
Za-methy)
more foo
than thej
of blood
pigs aver.
logue was
| by 0.409,
eight gain
zroups in-
mic, their
$ that of
d in their
ur, pallor,
>, Museu
sions and
morrhagic
ved upon
"appeared
to 4-day-
lete semi-
orbic acid
f calcium
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acid de-
ed symp-
pigs. Soft
sions and
re was no
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rbie acid

with the
atl. Insts.

th guinea

purified
ng differ-
)rackett)
ze weight
2d witha
th a diet
ight was
ilted ina
he maxi-
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ecessary.

Present
ompared
ment. In

March 1956

preliminary tests guinea pigs which have been
reared on a diet devoid of protein, but containing
14 amino acids proportioned to the amounts con-
tained in 20% Drackett protein, have grown
reasonably well over a period of 80 days with an
average daily gain in weight of 4.5 gm as compared
to a gain of 6 gm in the control animals.

1862. Comparison of metabolic energy con-
tributions of foods by rat growth under
conditions of energy restriction. E. E. Rice,
W. D. Warner,* P. E. Mone* anp C. E. Pot-
1nG.* Research Labs., Swift & Co., Chicago, Ill.
When fed diets which are restrictive in calories

but which are otherwise adequate for vigorous
growth, rats gain in proportion to calorie intake.
They consume basal and supplemented diets com-
pletely and show very uniform growth for each
level of supplementation, permitting accurate
comparisons after only 1 wk. of testing. The ex-
periments conducted indicate that short term
growth is equally affected by calories from carbo-
hydrate, fat or protein. The caloric availability of
aseries of 7 fats and fatty acids correlated closely
with the digestibilities of the fats as measured by
conventional intake-excretion studies, suggesting
that this technique may provide a convenient,
rapid method for measuring the caloric availa-
bility of various foods. It has been used success-
fully for this purpose with series of fresh and
processed fats.

1863. Relationship of vitamin Bs; to serum
protein and nonprotein nitrogen in the rat
during pregnancy. MarGaret L. Ross anp
Ruts L. PrKe (introduced by Mary L. Dopps).
Dept. of Foods and Nutrition, Pennsylvania
State Univ., University Park.

This study was designed to determine whether
the changes in total protein and nonprotein nitro-
gen levels in the serum of the rat during pregnancy
are similar to those reported for the human; and,
further, to determine the effects of a vitamin Bes
deficiency before and/or during pregnancy upon
the levels of serum protein and nonprotein nitro-
gen in the rat. One group of rats was subjected to
a depletion period prior to mating (Bs-deficient
diet plus 0.5 mg % desoxypyridoxine) and another
was maintained on laboratory chow until the day
mating was confirmed. On the first day of preg-
nancy each main group was sub-divided into 5 diet
groups and both the depleted and non-depleted
animals then received diets containing either 0.5
mg % desoxypyridoxine, or one of four levels of
pyridoxine (0, 0.4, 0.8, 1.2 mg %). Blood taken by
heart puncture at term was analyzed for total
protein, albumin, globulin and nonprotein nitro-
gen. The slight decrease in the concentration of
total protein, the significant increase in globulin
and reduction of nonprotein nitrogen in the serum

AMERICAN INSTITUTE OF NUTRITION

571

of the rat during pregnancy follow a trend similar
to that reported for the pregnant human. De-
pletion of maternal vitamin Bs, stores prior to
mating and/or pyridoxine deficiency during preg-
nancy lead to changes in serum protein and non-
protein nitrogen concentrations which are similar
to those reported for the toxemias of pregnancy in
the human.

1864. Effects of excess vitamin A palmitate
in paired feeding experiments on albino
rats. Myra M. Sampson, EstHER CARPENTER*
AND Rouanp Wiaeut.* Zoology Dept., Smith
College, Northampton, Mass.

Seven sets of 3 litter-mate female rats of the
Sprague-Dawley strain were used. Initial weights
varied from 126 to 150 gm and ages from 43 to 52
days. All rats were given a purified diet plus
powdered yeast and biweekly supplements of
vitamins A, C, D and E. The experimental rat in
each group was given approximately 25,000 ru of
vitamin A by mouth daily during the Ist wk. and
twice or three times that amount in succeeding
weeks. The pair-fed control was given the amount
of food eaten by the experimental rat on the previ-
ous day. The other control was allowed io eat ad
libitum. Each set was killed when the weight of
the excess A rat continued to fall. The first con-
sistent and progressive indication of hyper-
vitaminosis A was a reduction in efficiency of food
utilization which became apparent during the 2nd
week. The total ratio of weight gain to food intake
in one of the hypervitaminotic rats went as low as
13% of that of the intake-paired control. In ad-
dition to the usual symptoms of toxic hyper-
vitaminosis A, there should be mentioned a copious
yellowish discharge in the urethral region and a
marked thinning of the skeletal muscles. Changes
in the thyroid gland will be reported elsewhere.
(Supported in part by grants from the American
Cancer Society and the Massachusetts Branch of
the same.)

1865. Quantitative measurements of bone
density changes in rats fed diets of different
ealcium content. Haratp ScHRAER AND
RosEMARY SCHRAER (introduced by J. F. Mc-
CuiEenDoN). Radiology Dept., Albert Einstein
Med. Ctr., Northern Div., Philadelphia, Pa.
The degree of accuracy with which the mineral

content of bone can be measured has been mark-

edly improved by the development of a bone
density computing machine (Brown, Proc. Nail.

Electronics Conf. 5, 1949). Diaphyses of rat femurs

were removed, cleaned, dried and x-rayed with an

aluminum alloy wedge in juxtaposition. The films
were evaluated for x-ray mass and compared with

ash weights of corresponding bone samples. A

correlation ratio of .98 was obtained. Later, 30

weanling rats received a low calcium diet, and 20

572

received a diet adequate in calcium. The animals
were X-rayed with the wedge and killed, some at
36 days and thereafter at 7-day intervals for
several weeks. Rats fed Purina Dog Checkers were
used as controls. The films were evaluated for bone
density at the mid-point of the femur. Bone den-
sity values increased slowly on the low calcium
diet, and more rapidly on the control checker diet
and the diet adequate in calcium. After 64 days,
animals on the low calcium diet received a supple-
ment of calcium as calcium carbonate. Films of
animals on the supplemented diet showed a
marked increase in bone density values. This in-
crease was observed at 7 days by photodensi-
tometric measurements. The magnitude of density
increase observed indicated that the density
change could have been detected much sooner.
(Supported by grant No. A-602 (R) Natl. Insts. of
Health, PHS.)

1866. Torula yeast and liver necrosis. L. J.
ScHROEDER,* T. Hrratzka* anp A. H. Smita.
Depts. of Physiological Chemistry and Pathol-
ogy, Wayne Univ. College of Medicine, Detroit,
Mich.

In a preliminary study of the relation between
diet and liver necrosis, 4 groups of rats were given
a low-fat vitamin E-free diet, in which the protein-
nitrogen was supplied by casein, primary-grown
cerevisiae yeast, debittered brewer’s yeast or
tolula Yeast. All diets was isocaloric and contained
equal nitrogen. On these diets, the casein-fed rats
showed the best growth rate, the torula yeast-fed
animals the poorest. Gross and histopathologic
examination of the heart, lungs and spleen showed
no changes. Papillomas in the forestomach were
scribed to the vitamin E deficiency, since it was
seen in all diet groups. Many spontaneous deaths
occurred among the torula-fed animals; patho-
logical examination showed liver necrosis. Kidney
changes appeared to be associated with this
necrotic syndrome. A 2nd series of animals (90)
given the torula yeast diet showed 22% spontane-
ous deaths within 28 days; 60% of the animals died
by the end of the 5th week and 82% were dead at
42 days. All had developed liver necrosis. Torula-
fed animals killed from the 3rd to the 5th week
also showed this pathologic condition. Associated
kidney calcification appeared to be of a secondary
nature. The necrotic changes in the liver were seen
only in the torula yeast-fed rats; no necrosis was
observed when casein or cerevisiae yeast served as
the sole source of protein in these diets.

1867. Vitamin E in chick nutrition. M. L.
Scort anp T. S. Netson.* Cornell Univ., Ithaca,
N.Y.

Chicks fed a vitamin E-deficient diet (Poultry

Sci. 34: 1220, 1955) develop exudative diathesis

resulting in death by 4 wk. of age. These symptoms

FEDERATION PROCEEDINGS

Volume 16

were prevented by the addition of vitamin E or
substitution of dried brewers yeast for 7.5% and
10% torula yeast. Using this diet, studies were
conducted on the quantitative requirement of the
chick for vitamin E by feeding graded levels of
a-tocopherol and a-tocopheryl acetate. The
requirement was found to be approximately 10.10
vitamin E/lb, of diet. When DPPD was added to
the diet, the requirement was approximately 8 1u
vitamin E/lb. of diet. The vitamin E content of a
sample of alfalfa meal was determined chemically
by three different laboratories and found to be
approximately 73 mg d-a-tocopherol or 109 10 of
vitamin E/lb. Replacing torula yeast with 6.25%
and 12.5% alfalfa meal furnished, therefore, 5.8
and 11.6 1u vitamin E/Ib. of diet. The biological
activity of the alfalfa meal was compared with
that of 4.1 and 5.8 1u vitamin E/lb. of diet as
a-tocopheryl acetate, using White Plymouth
Rock chicks. The experiment showed that the
vitamin E activity of the alfalfa meal as deter-
mined by biological assay was approximately the
same as that determined by chemical analysis.

1868. Effect of vitamin B,,, aureomycin and
penicillin on growth of Guatemalan school
children. Nevin S. ScrimsHaw, Ovupu B.
TANDON* AND Carios P&reEz.* Inst. of Nutri-
tion of Central America and Panama, Guatemala,
C. A.

Significant effects were previously reported
(Federation Proc. 13: 1954) of daily oral adminis-
tration of 50 mg of aureomycin for 5 months on
growth of 72 rural school children compared with
73 receiving placebos. During a subsequent 25
months no significant differences have been en-
countered between experimental and control
groups. School children previously showing no
growth response to 50 mg of penicillin or penicillin
plus 20 ug of vitamin Biz daily for 24 months did
not respond to 1.5 mg of folic acid added for 12
additional months. Twenty-seven in the experi-
mental and 18 in the control group participated
for the entire period. For 18 months 228 school
children in four localities received 20 ug of vitamin
Bie daily or placebos; no differences in average
monthly gains in height and weight resulted from
the Bis. Pre-school children averaging 4.4 yr. were
given 20 ug of vitamin Bie or placebos. In 12
months, 43 receiving Biz showed an average bone
maturation gain of 8.6 months compared with 8.1
months for 46 controls. Tabulations completed for
the first 6 months show average monthly gains of
0.65 cm in height and of 0.17 kg in weight com-
pared with 0.70 cm and 0.16 kg for controls. It is
concluded that long-term oral administration of
vitamin By alone or the antibiotics aureomycin
and penicillin, even when Biz and folic acid are
added to the latter, has no significant positive or
negative effect on child growth in Guatemala.

lume 15

in E or
5% and
es were
t of the
avels of
2. The
ly 10.10
dded to
ely 8 1U
ent of a
mically
1 to be
19 1U of
n 6.25%
ore, 5.8
ological
2d with
diet as
ymouth
hat the
3 deter-
tely the
sis.

in and
school
pH B.
' Nutri-
utemala,

eported
dminis-
nths on
ed with
uent 25
een en-
control
ving no
snicillin
iths did
1 for 12
experi-
icipated
3 school
vitamin
average
ed from
yr. were
. In 2
ge bone
with 8.1
eted for
gains of
ht com-
ls. It is
ation of
eomycin
acid are
sitive or
mala.

March 1956

1869. Influence of vitamin E. deficiency in
rabbits on the incorporation of glycine-
1-C'* and formate-C' into glycine, serine
and methionine of skeletal muscle in vivo.
Joun T. Srue,* Dovetas E. Youne,* JAMES
8S. DinninG AND Paut L. Day. Dept. of Biochem-
istry, Univ. of Arkansas School of Medicine,
Little Rock.

Because increases have been found in the in-
corporation of glycine-1-C'* and formate-C™ into
the protein of skeletal muscle of vitamin E-de-
ficient rabbits over normal controls, it was of
interest to determine the effect of the deficiency
on the labeling of various amino acids of skeletal
muscle. Vitamin E-deficient and normal control
rabbits were injected with either glycine-1-C™
or formate-C" and killed 4 hr. later. The protein
of skeletal muscle was prepared and from it the
amino acids, glycine, serine and methionine were
isolated by ion exchange chromatography follow-
ing acid hydrolysis. When glycine-1-C'* was
injected there were only slight differences in either
glycine or serine specific activities between control
and deficient animals, with a tendency toward
higher values for the deficient. When formate-C™
was injected, little difference was found in glycine,
while the specific activity of the serine in the
deficient animals was increased over 100% above
the control. Methionine activity increased from
zero in the normal controls to a high activity in
the deficient rabbits.

1870. Urinary creatinine as a measure of en-
dogenous nitrogen. JANicE M. Smita, SHrH-
Dzune Cuen,* Epna Dicxk,* Auice Hareg,*
Martraa McMI.ian* anp Beuta V. McKey.*
(Dept. of Home Economics, Univ. of Illinois,
Urbana.

The creatinine nitrogen:endogenous nitrogen
for 7 men, 22-27 yr. of age, averaged 23.2+1.0
(S.E.). This was different statistically at the 5%
level from the ratio of 25.1+0.4 for 25 women pre-
viously reported from this laboratory (BRICKER
et al. J. Nutrition 44: 553, 1951). The validity of
using this ratio in lieu of endogenous values in
biological value estimations was further tested
using a food mixture containing 70% of cereal
protein. The mean biological value of the protein
mixture, calculated according to the Thomas
method, was 61.6+2.58 when the endogenous nitro-
gen was determined and 57.5+3.10 when the
endogenous nitrogen was estimated from the
CN/EN ratio. The estimated daily nitrogen re-
quirement for maintenance and adult growth
averaged 7.480.298 gm for the 7 subjects when
the nitrogen was supplied by the 70% cereal pro-
tein mixture. These findings were based on nitro-
gen balance data collected during 32 days of
controlled feeding divided into 3 periods as fol-
lows: Pd I, 12 days, low nitrogen diet; pd II, 10

AMERICAN INSTITUTE OF NUTRITION

573

days, 2.4 gm nitrogen daily; pd III, 10 days, 4.4
gm nitrogen daily.

1871. Lysine requirement of normal infant.
Setma E. Snyperman, L. Emmett Hott, Jr.,
Patricia Norton,* Dorotuy I. FowLer* anp
EILEEN HASSELMEYER.* Dept. of Pediatrics,
New York Univ. College of Medicine, New York
City.

The lysine requirement was determined for 6
normal male infants varying in age from 1 to 5
months. This was done by means of a synthetic
diet, the protein moiety of which was composed
of 18 L-amino acids given in the proportions found
in human milk. The lysine content of the diet was
reduced, being replaced by an equivalent amount
of nitrogen in the form of glycine, until the mini-
mal amount was ascertained which would support
normal weight gain, nitrogen retention and serum
protein concentration. All 6 of these infants re-
quired less than 90 mg of lysine (112 mg lysine
monohydrochloride) per kg per day. This quantity
is less than 3 the amount of lysine fed to infants
on commonly used cow’s milk formulas. When the
intake of lysine fell below the minimal level there
was an arrest of weight gain and a tendency for
serum protein values to drop; this was at the
expense of the serum albumin. Urinary excretion
of free amino acids was determined by means of a
Dowex-50 column, and showed a reduced excre-
tion of lysine. A comparison of the minimal re-
quirements of infants for lysine and tryptophane
indicates an L/T ratio of 3.0, a figure which com-
pares closely with ratios of 3.2 (Rose—adult men)
and 2.72 (Leverton—adult women). Our data fail
to provide evidence that a higher proportion of
lysine is required during the period of growth.

1872. Vitamin A and carotene serum levels in
depleted chicks and children in relation to
vitamin A or carotene intake. Rospert L.
Squiss, J. Epcar BrauHaM,* GuILLERMO
ARROYAVE* AND Nevin 8S. Scrimsuaw. Inst.
Agropecuario Nacional and Inst. de Nutricién
de Centro América y Panamé, Guatemala, C. A.
Blood serum vitamin A and carotene deter-

minations have proved revealing in nutrition

studies. Synthetic carotenes in cottonseed oil or
mixed with sugar lowered mortality and stimu-
lated growth when fed to provide 80-180 ug % in
five experiments of 5 wk. duration using 600 3-day-
old New Hampshire chicks depleted with a low
vitamin A basal ration. The serum levels of the
chicks receiving 180 ug % increased only 5 ug
vitamin A and 20 yg carotene/100 ml. The ca-
rotenoids from a dehydrated ramie (Boehmeria

nivea) meal, added at comparable levels, gave a

linear growth response and increased serum vita-

min A 8 ug and carotenoids 75 wg/100 ml. Three
local yellow corns fed for 35 days as 12% of the

574 FEDERATION PROCEEDINGS

basal ration increased serum vitamin A levels
from 2 wg to a maximum of 13 wg and serum
carotenoids from 23 yg to 167 »wg/100 ml. Upon
hospital admission, 10 children with Kwashiorkor
showed an average serum vitamin A level of 8
pug/100 ml. Administration of 1.5 mg of water
miscible vitamin A orally to these depleted chil-
dren resulted in marked elevation of vitamin A
serum levels to an average maximum of 52 wg/100
ml. in 1-2 wk. Average vitamin A values of 12 ug
and carotene value of 14 uwg/100 ml were found
in the sera of persons in a semi-famine area.

1873. Nature of supplementary value of vita-
min B, to proteins in the infant food,
Pablum (mixed cereal). BARNETT SURE.
Dept. of Agric. Chemistry, Univ. of Arkansas,
Fayetteville.

In previous communications it was suggested
that vitamin Bie supplements the proteins in
milled wheat flour, in presence of lysine and
threonine, by virtue of its possible function as an
activator or coenzyme of intestinal enzymes. In
attempting to improve the biological value of the
proteins in the infant food, Pablum (mixed cereal),
with amino acid additions, using the albino rat
as the experimental animal during a period of 6
wk., it was found that fortification of the basal
rations with 0.2% t-lysine, 0.2% pu-threonine, and
0.2% pu-methionine was followed by the same
gains of body weight and increase in protein
efficiency ratios as that obtained by additions of
0.2% u-lysine and 0.2% pu-methionine supple-
mented with 0.1 wg vitamin Bi2/animal/day. In
other words, vitamin Biz apparently produces a
sparing action on methionine requirements by
transmethylation.

1874. Protein nutrition of elderly women.
Peart P. Swanson, JANE LEICHSENRING,*
HELLEN LINKSWILER* AND Haze Fox.* Jowa,
Minnesota and Nebraska Exper. Stations, Ames,
St. Paul and Lincoln.

Day-by-day utilization of the nitrogen provided
by self-selected diets has been studied continu-
ously in 11_women for periods ranging from 49
days to more than 1 yr. The youngest subject
was 36 years of age; the oldest, 80. These long-
time balance studies suggest the occurrence of
three planes of protein nutrition among the indi-
viduals in this group of women; i.e. 1) previous
undernutrition as shown by continuous protein
deposition for long periods of time following
improvement of diet, 2) diminishing reserves of
body nitrogen during the periods of observation
on self-selected diets, and 3) equilibrium that is
the outcome of periods of positive and negative
retentions that occur in a cyclic manner. Cycles
of gain or loss of nitrogen are sometimes long,
sometimes short. However, the data indicated

Volume 15

that the diets of certain of these elderly subjects
apparently were not supplying enough protein
for maintenance of an excellent plane of nutrition,
even though the over-all picture of nitrogen
balance suggested that very little body tissue
was being lost. For example, some women studied
showed a tendency to retain extra protein when it
was added to their regular diets. As much as 3 of
the additional protein might be retained. The
effect was immediate and sustained for periods of
2 or 3 wk.

1875. Effect of aureomycin on anemia of the
gastrectomized rat. Marian E. SwENDSsEID,
Norma J. LoNG AND JAMES A. HALSTED (intro-
duced by W. H. Grirritu). Depts. of Home
Economics and Physiological Chemistry, Univ. of
California, and VA Ctr., Los Angeles.

Male Long-Evans rats weighing 150 gm were
gastrectomized according to the procedure of
Balfour (Proc. Staff Meet. Mayo Clin. 25: 434,
1950). They were placed on a semi-purified diet
containing all the vitamins except folic acid and
vitamin Bie. After 30 days, a moderate anemia was
present. Analysis of the liver tissue of these
animals 90 days after operation showed a great
reduction in both folic acid and vitamin B12 con-
tent as compared to the concentration of these
vitamins in liver tissue from unoperated litter
mate controls maintained on the same diet. Sup-
plementation of the diet with 200 y of folic acid
and 1 y vitamin B;2/100 gm singly or in combina-
tion had no effect on the anemia. Addition of
50 mg aureomycin/100 gm diet prevented the de-
velopment of anemia in either the presence or
absence of folic acid and vitamin Bie. Average
hemoglobin values for groups of gastrectomized
rats not receiving aureomycin ranged from 10.4
to 11.3 gm %; averages for gastrectomized rats
having aureomycin supplements ranged from
14.3 to 15.6 gm % hemoglobin. The latter values
were similar to those of control nonoperated rats.

1876. Lipid patterns: and atherogenesis in
cholesterol-fed chickens. Davin M. TEN-
NENT,* HENRY SIEGEL,* GUNTHER KuRon,*
WaLTHER H. Ort anp CHARLES W. MUSHETT.
Merck Inst. for Therapeutic Research, Rahway,
N. J., and Pathology Dept., Albert Einstein
College of Medicine, New York City.

In this investigation the results of plasma lipid
analysis have been correlated with the severity
of atherosclerosis in the aortae and _ brachio-
cephalic arteries of White Leghorn cockerels fed
a diet containing cholesterol and cottonseed oil
as described by Katz. The birds were raised to
8 wk. of age on standard starter ration and then
fed atherogenic diet until 16 wk. of age when they
were bled and killed. Four ml of blood taken from
each bird were used to prepare ACD plasma which

E XUM

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March 1956

was analyzed for total cholesterol and lipid phos-
phorus and fractionated by Cohn’s Method 10.
The amounts of cholesterol in Fractions I, II and
III, and in Fractions IV and V were determined.
The ‘a/a + 8’ and cholesterol/phospholipid ratios
were calculated. The results from 255 chickens
taken from 20 experiments conducted during the
past 2 yr. show a highly significant correlation
between the severity of atherosclerosis and the
lipid concentrations and ratios. The total choles-
terol values appear to be more important here than
in man. Presumably this is because the athero-
sclerosis in these chickens is predominantly of
dietary origin whereas in man other factors may
often be involved.

1877. Influence of diethylstilbestrol on growth
and reproduction of mink. H. F. Travis,*
C. F. Bassert,* R. G. WarRNER* AND J. K.
Loosur. U. S. Fur Animal Exper. Station and
Dept. of Animal Husbandry, Cornell Univ.,
Ithaca, N. Y.

Sixty female mink kits were weaned and divided
into 4 groups at 6 wk. of age and fed either 0, 0.2,
2.0 or 10 wg of diethylsilbestrol as a supplement
to a normal ranch ration. The 10-ug level sig-
nificantly (P < 0.05) reduced the body length
and weight gains of the minks during a 23-wk.
experimental period. Mean increases in body
length for the 4 treatments were 5.6, 5.6, 5.5 and
5.1 inches; respectively. Mean increases in body
weight were 1.40, 1.33, 1.34 and 1.17 lb., respec-
tively. There were no differences in body gain
until after the 10th wk. of treatment. Forty adult
females were divided into 4 groups and fed 0,
0.2, 1.5 or 10.0 ug of diethylstilbestrol as a supple-
ment to their ration from approximately 1 month
before to 2 months after the mating season began.
The 10-ug level significantly reduced (P < 0.05)
the number of kits born, when compared to the
other treatments. Average number of kits per
female were 3.2, 3.9, 5.9 and 0.9 for the different
levels, respectively. The two lower levels of di-
ethylstilbestrol did not significantly affect the
number of kits.

1878. Effect of fructose feeding on glucose
tolerance in man. THEODORE B. vAN ITALLIE,
Kenneth H. SHutt anp Mary B. McCann
(introduced by F. J. Stare). Dept. of Nutrition,
Harvard School of Public Health, Boston, Mass.
Hill, Baker and Chaikoff (J. Biol. Chem. 209:

705, 1954) have reported that normal rats fed

an adequate diet containing fructose as the sole

carbohydrate display a loss of glucose tolerance.

Hill et al. ascribed the diminished capacity of

fructose-fed rats to utilize glucose to an impair-

ment of the hepatic glucokinase system caused by
prolonged fructose feeding. They found that the
glucose tolerance curves in fructose-fed rats re-

AMERICAN INSTITUTE OF NUTRITION

575

sembled those observed in fasted rats. To deter-
mine whether the same phenomenon occurs in
man, glucose tolerance was studied in normal
subjects before and after they had consumed a diet
containing fructose as the sole source of carbo-
hydrate for 5 days. The diet was adequate in
calories and protein and provided approximately
250 gm carbohydrate/day, all as fructose. The
effect of the same diet with all the carbohydrate
omitted on glucose tolerance in some of the same
subjects also was determined. When all carbo-
hydrate was omitted from the diet for 3 days,
significant impairment of glucose tolerance was
observed in every case. Studies of peripheral
capillary-venous glucose differences indicated that
a diminished rate of extrahepatic glucose uptake
was present. By contrast, when fructose was in
the diet as the sole source of carbohydrate for 5
days no impairment of glucose tolerance of the
kind reported by Hill et al. for rats was observed.
Capillary-venous glucose differences also re-
mained normal after fructose feeding.

1879. Interrelation of protein and calories
during caloric restriction. J. J. VITALE,*
P. L. Wurre,* L. Stntsterra,* D. M. Heestep
AND N. ZAaMCHECK.* Dept. of Nutrition, Harvard
School of Public Health and Dept. of Pathology,
Boston Univ. Med. School, Boston, Mass.
The present work was undertaken to study the

physiological response of animals fed sub-main-

tenance calories at varying levels of protein as
measured by weight loss, tissue enzyme activity

(xanthine oxidase of liver and duodenal mucosa,

succinic oxidase of liver and alkaline phosphatase

of liver, duodenal mucosa and serum) and morpho-
logical change. Rats (300 gm body wt.) were fed

5 gm (20 cal.) a day of diets containing 10, 20 and

40% protein. Although the 40% protein group

lost slightly more weight and the mortality rate

was higher than in other groups, all animals lost
weight rapidly and no difference in nitrogen
balance was noted through 26 days. In an experi-
ment in which 9 gm (36 cal.) of diet (5, 10, 20 and

40% protein) were fed per day, the 5% protein

group lost weight the least rapidly during 42

days. Since higher levels of protein seemed detri-

mental, the effect of protein on weight loss and
mortality was studied. Groups of animals fed 5 gm

(20 cal.) of diets/day containing 0, 5, 10, 20 or

40% protein had mortality rates of 0, 25, 25, 33

and 66% at the end of 10 days and 8, 50, 50, 83

and 100% in 12 days, respectively. Under the

degree of caloric restriction studied, the higher
protein diets appeared to be detrimental. Tissue
enzyme activity and morphology will be reported.

1880. Species differences in excretion of intra-
venously injected calcium®. Rosert L.
WANNER,* JOAN R. Moor,* FeEttx BRONNER,*

576 FEDERATION PROCEEDINGS

Nancy S. Pearson* anp Ropert 8S. Harris.
Dept. of Food Technology, Massachusetts Inst.
of Technology, Cambridge.

The excretion of Ca‘*® in the urine and feces
during 5 days following intravenous injection of
Ca** was studied in 9 boys (12-15 yr.), one young
man (21 yr.), 3 adult rhesus monkeys and 10 albino
rats (120 days old). The results of these analyses,
together with the results of a similar study by
Maletskos (Ph.D. thesis: Massachusetts Inst.
of Technology, 1955) on 8 adult dogs are pre-
sented below:

% Dose Ratio Fecal/
pe Ca’ Excreted Urinary Ca‘

Species Injected (0-5 days) Excreted
Human, boys 0.7 8 1:2
Human, adult 3.4 20 1:2
Monkey, adult ef 9 1:2
Rat, adult 1.0 14 22:1
Dog, adult 1.5 42 10:1

The excretion of intravenously injected Ca*®
by the rhesus monkey resembles that by human
beings; that by rats and dogs is quite different.
These data indicate that the endogenous excretion
of calcium by human beings and monkeys is simi-
lar, and unlike that of dogs and rats.

1881. Influence of amino acids and other
organic compounds on_ gastrointestinal
absorption of radiocalcium and radiostron-
tium. R. H. Wasserman,* C. L. CoMarR AND
Max M. Notp.* Med. Div., Oak Ridge Inst. of
Nuclear Studies, Oak Ridge, Tenn.

Various organic compounds, notably the es-
sential amino acids and some nonessential amino
acids, were examined as to their effect on the
gastrointestinal absorption of Ca*® and Sr® in
the rat. Fasted young albino male rats were orally
dosed with a solution containing Sr®*, Ca**, 10 mg
carrier CaCl. and 0.84 mm of the test substance.
Twenty hours after dosage, the animals were
killed, the femurs removed and assayed by stand-
ard procedures. The radioactivity found in the
bone at this time was used as a measure of mineral
absorption. The results may be summarized by
listing the substances tested in decreasing order
of response: Series A: ut-lysine = L-arginine >
L-tryptophane > t-leucine > t-histidine > L-
isoleucine > t-methionine = t-valine =>
L-threonine + t-phenylalanine = control. Series
B: t-lysine > t-aspartic acid > L-glutamic acid
hydroxy-t-proline > pu-tyrosine > L-serine
glycine > t-alanine = t-proline = control.
Series C: lactose > u-lysine = L-arginine > L-
leucine = gluconate > t-methionine > lactate >

citrate = vitamin B mixture = control. As an
indication of magnitude of response, the lysine
approximately doubled the bone uptake of the

radioisotopes. When the radioisotopes were in-

IR i

Volume 15

jected instead of administered orally, lysine
treatment did not increase the amount of Ca‘*®
and Sr®* in the bones. This indicates that the
foregoing results reflect gastrointestinal absorp-
tion rather than bone mineralization. In regard to
the comparative metabolism of calcium and
strontium, it was observed that Ca*® was prefer-
entially absorbed from the gut by a factor of 1.6
over Sr®*, and substances that promoted Ca‘
absorption were generally slightly more effective
in promoting Sr*® absorption. These data suggest
that these two minerals are to some extent inde-
pendently absorbed and probably compete for the
same site in the transport process across the
gastrointestinal barrier.

1882. Vitamin E deficiency and tricarboxylic
acid cycle oxidation: effect of pyrophos-
phate and adenine nucleotide concentra-
tion. I. M. Wernstocx,* A. D. GoLpRicH* AND
A. T. MitHorat. Depts. of Psychiatry and Medi-
cine, Cornell Univ. Med. College, New York
Hosp., New York City.

During vitamin E deficiency, oxidation of tri-
carboxylic acid cycle substrates by washed liver
homogenates supplemented with 2 X 10°? m ATP
is greater than controls. Replacement of the ATP
with ADP or AMP increased the oxygen consump-
tion of controls but had no significant effect on
oxidation by the deficient group (WEINSTOCK
et al. Arch. Biochem. Biophys. 57: 496, 1955). Pres-
ent investigations show that the addition of
increasing concentrations of pyrophosphate
caused a significant reduction in oxygen consump-
tion by vitamin E-deficient systems without
markedly affecting the ATP-supplemented con-
trols. Pyrophosphate also inhibited oxidation by
ADP- and AMP-supplemented control and de-
ficient systems. The difference in oxygen
consumption between the 2 X 10-? m ATP-supple-
mented control and vitamin E-deficient liver
preparations could also be reduced by increasing
the concentration of the ATP supplement. In-
creasing concentrations of ATP caused a marked
reduction in oxidation by deficient systems with-
out significantly affecting the controls. Higher
concentrations of ADP also decreased oxygen
consumption as compared to 2 X 10-3 m ADP-
supplemented systems. Formation of pyrophos-
phate from ATP by these liver preparations could
not be detected and there was no change in
pyrophosphatase activity as a result of the
deficiency.

1883. Iron administration in a tropical area.
P. L. Wuite,* A. Qurroz,* L. Gonzauas,* 8.
Moratss,* J. ATxins,* M. F. Trutson,* C.
Cotiazos* anp D. M. Heestep. Internatl. Co-
operation Admin., Dept. de Nutricion, Inst.
Nacional de Higiene and Unidad Sanitaria

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March 1956

Loreto, Ministerio de Salud Publica, Lima Peru;
Dept. of Nutrition, Harvard School of Public
Health, Boston, Mass.

The effect of various levels of iron administra-
tion upon. the anemia in a tropical area where
infestations of hookworm and other parasites are
frequent has been studied. School girls, ages 6-12,
in the city of Iquitos, Peru, were subjects. The
hemoglobin levels ranged from 5 to 12 gm %,
average 9.78, and the severity of the anima was
proportional to the hookworm load. The average
daily iron intake was estimated as approximately
12 mg. Four groups of about 50 children received
0 (placebos), 15, 30 and 50 mg of iron as ferrous
sulfate daily for periods up to 14 wk. The response
was essentially maximal within 6 wk. and all
levels of iron appeared equally effective at this
time. The most anemic children :aowed hemo-
globin increases of 3-4 gm %. Hemoglobin level: of
10-12 gm were not increased. When iron adminis-
tration was stopped, the hemoglobin levels of the
50-mg group were maintained for 3 months but
returned to pre-therapy levels by 8 months. Ap-
parently 15 mg or less of extra dietary iron will
protect or correct the frank anemia associated
with the usual levels of parasitism in the area
studied.

1884. Effect of intake of linoleic acid on un-
saturated fatty acids of serum of infants.
Hitpa F. Wiese, Arttp E. Hansen, Doris
J. D. Apam* anp MarsorreE A. BauGHan.*
Dept. of Pediatrics, Univ. of Texas Med. Branch,
Galveston.

Nine infants were observed for 66 dietary
periods of 7 days each. All infants were given a
diet low in linoleic acid (0.04-0.5% of the total
calories). The dietary intake of linoleic acid was
then varied from 1 to 8% of the calories by sup-
plementation with the esters or the triglycerides.
Di-, tri- and tetraenoic acids in the fasted blood
serum were determined spectrographically. At
a 1% caloric level of linoleic acid given as methyl
or ethyl esters with skimmed milk there were no
significant changes in the serum levels for 2, 3
and 4 double bond fatty acids from those found
on the low fat diet. In contrast, supplementation
with trilinolein resulted in significant increases
in both di- and tetraenoic acids. These latter
values were of the same order of magnitude as
when the infants received an evaporated milk
mixture containing about 1% of the calories as
linoleic acid. At a 3% caloric level of linoleic acid,
as the methyl ester with skimmed milk, responses
in di- and tetraenoic acids in the serum were
similar to those for 1% trilinolein or evaporated
milk. When 3% of the calories as linoleic acid was
supplied in the form of trilinolein with skimmed
milk, serum levels of di- and tetraenoic acids rose

AMERICAN INSTITUTE OF NUTRITION 577

significantly higher than with 3% M-linoleate or
evaporated milk. These serum values were higher
even than when 5% caloric linoleic acid as E-
linoleate was fed. When half skimmed milk (15%
cal. fat, 0.5% linoleic) was supplemented with
E-linoleate (1-8%), dienoic acid in the serum
increased to values above those found when sup-
plementation was with skimmed milk (1.4% cal.
fat, 0.04% linoleic). When evaporated milk (39%
cal. fat, 0.9% linoleic) was supplemented with
esters of linoleic acid, serum levels for dienoic
acid were higher than with skimmed milk but
differed little from those with half skimmed milk.
There was no enhanced effect for trilinolein at
1-8% caloric intakes when half skimmed milk
was substituted for skimmed milk. Changes in
serum content for tri- and tetraenoic acids oc-
curred at a slower rate than for dienoic acid,
particularly when linoleic acid was given in ester
form and the intake of calories from fat was low.
(Supported in part by the USDA through a con-
tract sponsored by the Human Nutrition Research
Branch, Agric. Research Service.)

1885. Nutritive value of glycine-related com-
pounds for the chick. Rospert L. Wixom,*
. GeorcE E. Prpxin* anv Paut L. Day. Dept. of

Biochemistry, School of Medicine, Univ. of Arkan-

sas, Little Rock.

Since the addition of serine to a glycine-deficient
ration improved the growth rate of chicks (Wixom
et al., J. Nutrition 56: 409, 1955), it was desirable
to test other compounds to see how specific glycine
was in stimulating chick growth. Each substance
tested was added at a level equimolar with 1.5%
glycine to a basal diet containing 35% casein,
arginine, cystine and the other usual components.
In addition to the earlier reported diammonium
citrate, 1.78% pu-alanine or 2.94% t-glutamic acid
were found to be ineffective under experimental
conditions where 1.50% glycine stimulated extra
growth. While the methylated glycines are effec-
tive precursors of glycine for the rat, it was found
that the addition of 2.34% betaine, 2.06%
dimethylglycine, or 1.78% sarcosine to the basal
diet gave a growth rate that was essentially the
same as that of the basal group. Apparently, then,
the substances reported to be effective as sub-
stitutes for glycine are limited to serine and
creatine. The addition of both 1.50% glycine and
2.10% pu-serine to the basal diet did not improve,
and actually lowered, the growth rate of chicks in
comparison with those receiving only 1.50%
glycine as a supplement. These data may be in-
terpreted as showing the possibility of an in vivo
synthesis of glycine from serine or vice versa.

1886. Tocopherol nutriture in a patient with
xanthomatous biliary cirrhosis. Catvin W.
Wooprurr. Dept. of Pediatrics and Div. of

578

Nutrition, Vanderbilt Univ. School of Medicine,

Nashville, Tenn.

A 42-yr.-old white woman who had xanthom-
atous biliary cirrhosis of 5 yr. duration and who
had been ingesting a low fat diet for over 3 yr.
had no tocopherol in her serum. Creatinuria and
pentosuria were also present. A severe defect in
fat absorption was demonstrated. The adminis-
tration of 600 mg of a-tocopherol by mouth was
not followed by its appearance in the blood. After
3 months of therapy with 100 mg of a-tocopherol
emulsified with Tween 80 daily, the creatinuria
and pentosuria disappeared. After 6 months the
serum tocopherol concentration was 0.35/100 ml.
Creatinuria, pentosuria and negligible serum
tocopherol concentrations recurred in 9 months
after tocopherol administration was discontinued.
Increased susceptibility of the red blood cells to
hydrogen peroxide hemolysis and a very low sub-
cutaneous tissue tocopherol content were found.
Treatment with a water soluble preparation of
a-tocopherol was again associated with disap-
pearance of creatinuria and the appearance of
tocopherol in the serum. These biochemical find-
ings suggest that this patient had a relative
deficiency of vitamin E secondary to a severe,
long-standing defect in fat absorption.

1887. Choline compounds and ethylene oxide
treatment of diets. R. 8S. Yamamoto, E. A.
Hawk AND QO. MICKELSEN (introduced by J. G.
Bieri). Natl. Insts. of Health, Bethesda, Md.
Studies of purified diets exposed to gaseous

ethylene oxide (HAWK AND MICKELSEN, Science

121: 442, 1955) showed that rats fed treated diets

containing choline dihydrogen citrate or choline

bitartrate survived, while those fed diets con-
taining choline chloride died within 4-5 wk. Ex-

posure of the purified diets to ethylene oxide did

not a) result in any destruction of choline as

measured by chemical tests and b) did not change
the mortality or growth response when sodium
citrate or citric acid equivalent to the choline
citrate were added to the choline chloride diet,
but c) resulted in reduced mortality and improved
growth when 1% citric acid was present in the
choline chloride diet. When the B vitamin mixture
containing choline chloride was treated with
ethylene oxide and then incorporated in the
purified diet, growth rate and mortality of
weanling rats were the same as those on the diet
that had been exposed in toto. Similar results were
obtained when choline was omitted during treat-
ment of the whole diet. However, weanling rats
grew as well as those fed untreated diet if the
vitamin mixture minus choline was first treated
and incorporated together with choline in the diet.

FEDERATION PROCEEDINGS

Volume 16

The present evidence indicates that choline
chloride or some other ingredient in the purified
diet must be present during ethylene oxide treat-
ment for destruction of the B vitamins.

1888. Effect of coenzyme A precursors on
hepatic coenzyme A levels in pantothenic
acid-deficient and organic sulfur-deficient
rats. CHING-SING YANG,* BEVERLY STEWART*
AND Roxpert E. Ouson. Dept. of Biochemistry
and Nutrition, Grad. School of Public Health,
Univ. of Pittsburgh, Pittsburgh, Pa.

It has been observed (OLson R. E., Federation
Proc. 12: 252, 1953) that dietary restriction of the
sulfur amino acids may depress hepatic coenzyme
A levels in the rat as much as the dietary restric-
tion of pantothenic acid (OLson, R. E. ann N. O.
Kaptan, J. Biol. Chem. 175: 515, 1948). It is ap-
parent from these studies and others (HOAGLAND,
M. B. anp G. D. Novetu, J. Biol. Chem. 207:
767, 1954) that both pantothenate and cystine or
methionine are required by the rat for the bio-
synthesis of coenzyme A. Studies in this labora-
tory have also shown that supplementation of the
diets of sulfur-deficient rats with cystine results
in a much more sluggish hepatic coenzyme A
synthesis than supplementation of pantothenate-
deficient rats with pantothenate. To explore this
phenomenon further, pantothenic acid-deficient
and sulfur amino acid-deficient rats were given
pantothenate, pantetheine, pantethine, N(d-
pantothenyl-l-cysteine), and N (d-pantotheny]-l-
cystine) in molar equivalent doses and coenzyme
A content of their livers determined at the end
of the lst day. The pantothenic acid-deficient
animals responded to all compounds with increases
in hepatic coenzyme A although pantothenate
itself was most effective. In the animals deprived
of organic sulfur, however, none of these com-
pounds was effective. The data suggest that
synthesis of hepatic coenzyme A occurs de novo
from nutrients and other small molecules and that
the capacity for this synthesis is impaired in sulfur
amino acid-deficiency in the rat. (Supported in
part by the Nutrition Fndn. and the Re-
search Corp.)

1889. Factors influencing amino acid utiliza-
tion in adult male albino rat. SxHrAne P.
Yana* anpD Peart P. Swanson. Home Eco-
nomics Research Dept., Towa State College, Ames.
A mixture of essential amino acids simulating

the acids occurring in an intact protein may not

serve as an effective source of nitrogen for the
maintenance of nitrogen equilibrium in the adult
male albino rat. This is true of the essential amino
acids present in lactalbumin. However, a modifica-
tion of the mixture within the nitrogen framework

ume 15

-holine
urified
treat-

rs on
thenic
ficient
WART*
omistry
Health,

eration
of the
nzyme
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GLAND,
m. 207:
tine or
he bio-
labora-
1 of the
results
yme A
henate-
pre this
eficient
> given

N(d-
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enzyme
she end
eficient
creases
thenate
eprived
e com-
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le novo
nd that
n sulfur
rted in
he Re-

itiliza-
ANG P.
ne Eco-
, Ames.
ulating
nay not
for the
1e adult
(| amino
\odifica-
mework

March 1956

of a 4% lactalbumin diet makes it biologically
efficient in this respect when fed, either alone or
with nonessential acids, in amounts providing
total nitrogen equivalent to the lactalbumin diet.
The influence of the incorporation of different
dietary carbohydrates and the effect of varying
the energy intake on the nitrogen turnover when
this mixture is fed have been studied also. Of
the 4 carbohydrates tested, i.e., dextrin, starch,
dextrose and sucrose, only rats ingesting dextrose
passed into acute negative nitrogen balance. The

—rr se 7

AMERICAN INSTITUTE OF NUTRITION

579

energy provided by the day’s food also influenced
the retention of nitrogen markedly. For example,
when the daily ration provided 66 cal., the average
nitrogen balance for a 7-day period was +86 mg;
when it furnished 37 cal., the balance was —164
mg. When the daily energy value of the ration
approximated that of the lactalbumin diet con-
sumed ad libitum, the retention was +17 mg. In
this case the energy value appears to be approach-
ing a critical level, because 50% of the balances
were negative.

AMERICAN ASSOCIATION OF IMMUNOLOGISTS

Fortieth Annual Meeting
Atiantic City, New Jersey, Aprit 16-20, 1956

An asterisk * following an author’s name indicates “by invitation”

1890. Increased susceptibility of mice to bac-
terial endotoxins induced by pertussis
vaccine. Rospert 8. ABERNATHY* AND WESLEY
W. Spink. Dept. of Medicine, Univ. of Minne-
sota Med. School, Minneapolis.

In selected strains of mice, injections of per-
tussis vaccine cause increased sensitivity to the
lethal action of histamine, anaphylaxis and bac-
terial vaccines. The present experiments show
altered reactivity to bacterial endotoxins follow-
ing pertussis vaccine. While female ABC mice
revealed no increase in sensitivity to histamine
following pertussis vaccine, female Swiss-Webster
mice showed a 200-fold increase. But both ABC
and Swiss-Webster mice receiving pertussis vac-
cine were more susceptible to the endotoxins of
Brucella melitensis and of Salmonella typhosa.
The disparity between histamine and endotoxin
susceptibility in the ABC strain makes it doubtful
that the altered reactivity to endotoxin is medi-
ated through a histamine-releasing mechanism.
Both 9-alpha-fluorohydrocortisone and chlorpro-
mazine protected normal Swiss-Webster mice
against brucella endotoxin, but only the steroid
offered protection against endotoxin in mice given
pertussis vaccine.

1891. Persistence of poliomyelitis virus dur-
ing serial passage of HeLa cells. W. WiLBUR
ACKERMANN. Dept. of Epidemiology and Virus
Lab., Univ. of Michigan, School of Public Health,
Ann Arbor.

When a culture of HeLa cells is exposed to
poliomyelitis virus (under proper environmental
circumstances), a portion of the culture (94%) is
destroyed and a portion survives. The cells which
survive are altered by the experience and develop
new properties and yet they retain their viability.
The object of this report is to characterize the
cultures which arise from such altered cells. The
findings are: 1) The cultures are easily distin-
guished from standard lines of HeLa cells by mor-
phology. 2) While readily susceptible to superin-
fection by all types of poliomyelitis viruses, they
are resistant to the cytopathogenic effect of the

superinfecting agents. Growth curves of type III
poliomyelitis virus in type II carrier cells indicate
that multiplication of virus proceeds more slowly
than in standard lines of cells. 3) The cells will
undergo sustained subcultivation (30-40 passages)
with retention of their peculiar properties. 4)
However, for sustained multiplication and
stability, the medium must contain specific im-
mune serum corresponding to the immunologic
type of virus used to produce the culture. 5) When
immune serum is withdrawn from a culture, the
cells will despoi] themselves. Under such condi-
tions the cells may give rise to virus of the original
infecting type. This capacity is retained despite
prolonged serial passage of the cells in immune
serum. 6) Though the data indicate that the virus
persists and undergoes limited multiplication in
the culture through repeated passage, it is not
detectable by virtue of its infectivity in each
passage. 7) Collectively, the data support the view
that this host-virus relationship is a cellular rather
than an ecological or cultural phenomenon.

1892. Antigenic components of immune sera
present in specific precipitates. FRANK L.
ApLeR. Div. of Applied Immunology, Public
Health Research Inst. of The City of New York,
Inc. New York.

Guinea pigs were injected with 8-10 times
washed precipitates (0.15-0.30 mg N) formed by
the addition of guinea pig serum to rabbit antisera
against guinea pig serum. By agar diffusion meth-
ods it was shown that antibodies against 5 or more
distinct components of rabbit serum were gener-
ally produced. One of these antigens was present
in maximal concentration in the gamma-globulin
fraction, though it was electrophoretically heterog-
enous; the other antigens were alpha- and/or
beta-globulins. Absorption of the cavian immune
sera with sheep red cell stromata, sensitized with
rabbit antiserum and washed thoroughly, caused
reduction or removal of antibody against all these
antigens. When rabbit antiserum against bovine
serum albumin (BSA) was subjected to electro-
phoresis in agar, and cavian antiserum of the type

580

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ANK L.
Public
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times
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caused
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bovine
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he type

March 1956

described was employed to precipitate the antigens
of rabbit serum (immuno-electrophoresis), only
one of the several bands of precipitate formed
could be stained by the subsequent addition of
fluorescein-labeled BSA. Thus all detectable
rabbit antibody against BSA appeared to be
associated with one of the antigenic components
of rabbit serum. This antigen was the electro-
phoretically heterogenous component mentioned
above.

1893. Action of plague toxin on diphospho-
pyridine nucleotide. Samuet J. AJL, JAMES
Rust, JR., JEANETTE WOEBKE AND DONALD
H. Hunter (introduced by JosepH E. SMADEL).
Dept. of Bacteriology, Div. of Communicable
Diseases, Walter Reed Army Inst. of Research,
Washington, D. C.

Diphosphopyridine nucleotide (DPN) is split by
purified plague toxin into at least 2 components,
one of which is nicotinamide mononucleotide.
The reaction is phosphate dependent and proceeds
in an enzymatic manner. The equilibrium of this
system is established when approximately 30% of
DPN is affected. The addition of specific antibody
or nicotinamide, a well known DPNase inhibitor,
inhibits the reaction. A close correlation exists
between the toxicity of a preparation for mice and
its ability to destroy DPN in vitro. Thus, when
toxin is either heated, toxoided with formalin,
hydrolyzed or treated in any fashion so as to
render it atoxic for mice it also loses its ability to
break down DPN. Mice injected with a few LD50’s
of plague toxin develop a small but statistically
significant reduction in DPN levels of their eryth-
rocytes below the normal range. This finding is
specific for plague toxin since other bacterial
toxins (Salmonella typhimurium, Escherichia coli)
failed to cause a similar effect in moribund mice.

1894. Nature of first component of comple-
ment. E. L. Brecker. Div. of Immunology,
Walter Reed Army Inst. of Research, Wash-
ington, D. C.

Sensitized sheep red cells containing the first
(C’1), the second (C’2) and the fourth components
(C’4) of guinea pig complement (EaC’1,4,2 pre-
pared according to the method of Levine et al.)
have been found capable of hydrolyzing tosyl
arginine methyl ester (TAME) at a significant
rate. Sensitized sheep cells (Ea) are not capable of
such hydrolysis. Under the same circumstances
acetyl tryptophan ethyl ester is not hydrolyzed by
either EaC’1,4,2 or Ea. The general technique
used was to incubate 5 X 10° cells/ml at 40° in 0.15
M NaCl, 0.001 m Ca, 0.01 m in TAME buffered with
0.02 m trishydroxymethyl amino methane at pH
7.8. The degree of hydrolysis was measured by the
amount of acid produced by the substrate with
EaC’1,4,2 less the amount produced on

AMERICAN ASSOCIATION OF IMMUNOLOGISTS

581

incubation with Ea. This latter was not any
greater than that produced by the incubation of
TAME alone. Under these circumstances, al-
though C’2 activity is destroyed within the first
+ hr., the rate of hydrolysis is linear with time for
over 6 hr. When the C’l on the complemented
cells is specifically inactivated by di-isopropyl-
fluorophosphate (DFP), a specific inhibitor of
esterases, the ability to hydrolyze TAME is lost.
The C4 activity of the DFP-treated cells is re-
tained. It is concluded that C’l is a protease with
esterase activity capable of hydrolyzing TAME.

1895. Possible multiplicity reactivation of
‘damaged’ poliovirus particles to yield
infectious virus. Francis L. Biack* AND
JosepH L. Metnicx. Section of Preventive
Medicine, Yale Univ., New Haven, Conn.

This investigation is concerned with multi-
plicity reactivation as a possible source of residual
infectivity in poliovirus preparations treated with
various inactivating agents. Virus clones were
isolated by the plaque technique from prepara-
tions that had been partially inactivated by elec-
tron bombardment or by formalinization. These
clones were then characterized serologically. The
plaques derived from untreated or lightly bom-
barded material were found to yield a smaller
fraction (5%) of ditypic isolates than did those
derived from heavily bombarded (1 to 2 million
rep) material (15%). The ditypic isolates in the
unirradiated control material may have arisen as
a result of virus aggregation or limitations in the
isolation technique. It is suggested that the
increased number of ditypic isolates in the irradi-
ated samples may have been largely due to multi-
plicity reactivation where more than one type were
involved. If this was the case, the data indicate
that the recombinants arising as a result of this
reactivation we.e unstable, and resegregated into
the original types during further multiplication.

1896. Rate of localization of antirat-kidney
antibodies. Monte Biav,* EuGene D. Day*
AND Davip PRESSMAN. Roswell Park Memorial
Inst., Buffalo, N. Y.

It has been shown previously that injected
antikidney antibodies are localized rapidly. The
present st’ dy was made to measure quantitatively
the rate of removal of antibodies from the circula-
tion. The globulin fraction of rabbit antirat-
kidney antiserum was radioiodinated and injected
intravenously into rats. After circulating for vary-
ing periods of time, the plasma was removed and
reinjected into other rats for assay of residual
localizing activity. The data indicate at least 2
different kidney localizing components in the
antiserum. The antikidney activity disappears
from the blood with a half-time of 7 min. until
70-75% is removed, at which point the rate of

582

removal decreases sharply to a 90-min. half-time.
The half-time of the major component is that ex-
pected for complete removal on one passage
through the kidney, calculated on the basis of
published values for blood volume and kidney blood
flow. This indicates that the antigens responsible
for localization are in intimate contact with
blood. The decrease of liver localizing activity
has a similar 2-component pattern with half-times
of 4 min. and 30 min., respectively. The antiserum
produced against rat kidney has some cross
reaction with liver. That the major portion is not
cross-reacting, however, was shown by passive
transfer through nephrectomized rats. After 30
min. in normal rats, 75% of the original kidney
activity and 85% of the liver activity were re-
moved. After 30 min. in nephrectomized rats, 85%
of the liver activity was removed, but only 40% of
the kidney localizing activity.

1897. Microscopic observations of acute
effects of anti-red cell sera on the extra-
and intrahepatic circulation (motion pic-
ture). Epwarp H. Buoce aNnp CARLO PIOVELLA
(introduced by I. H. Lerow). Dept. of Anat-
omy, Western Reserve Univ., Cleveland, Ohio.
Rabbit sera to frog and rat erythrocytes with

agglutinating and hemolytic titers of 1:1280 and
1:5000, respectively, were injected into the hepatic
portal system of these animals. The visible reac-
tions to these sera within the circulation were
studied microscopically in vivo in tissues trans-
illuminated with the quartz-rod technique. The
extrahepatic reactions are illustrated in the
abdominal veins of frogs where erythrocyte aggre-
gates as large as 800 » form the instant the sera
enters the circulation. Such aggregates immedi-
ately reduce the flow and occasionally plug these
vessels. The intrahepatic reactions are essentially
similar in the frog and rat. When the erythrocyte
aggregates enter the portal venules these vessels
often dilate and occasionally plug, temporarily.
The principal reactions occur in the sinusoids.
The aggregates are stored, due to the closing of the
sinusoids’ outlet sphincters, and concentrate due
to the Ayperpermeability of these vessels. How-
ever, phagocytosis of the aggregates by the
Kupffer cells is not seen. Concomitant with these
reactions in the liver a progressive peripheral
anemia occurs. Jn vitro hetero-agglutination of
frog and rat cells is seen as well as a reduction in
the red cell count, reticulocytosis, spherocytosis
and poikilocytosis.

1898. Bacterial toxins: toxins of pathogenic
Escherichia coli. DANiEL A. BoROoFF AND
Franco Rrnautpi.* New England Inst. for Med.
Research, Ridgefield, Conn., and Galesburg State
Research Hosp., Galesburg, Ill.

Pathogenic Escherichia coli, according to

FEDERATION PROCEEDINGS

Volume 1§

Vincent (Comp. rend. 1624, 1925) elaborate 2
toxins: one, thermostable enterotoxin, and the
other, a thermolabile neurotoxin found in the
culture medium after 5 days’ incubation. Baruk
(J. Ment. & Nerv. Dis. 110: 218, 1949) is of the
opinion that the latter toxin affects the central
nervous system and is responsible for certain
mental disorders. In the course of our study of this
neurotoxin, animals were injected subcutaneously
and intravenously with this substance. Within 2
hr. after the injection the animals became passive,
lost interest in their surroundings and could be
placed in various unnatural positions. However,
when the animals were put in danger of imminent
fall, they would move to regain balance. The ani-
mals also moved when some external force was
applied but, as soon as the force was withdrawn,
at once relapsed into passivity. Muscle tonus and
the righting reflex were always present. This state
resembled in many respects that described by De
Jong (Phys., Pharm., Microbiol. 1: 4, 1931) in his
study of bulbocapnine catatonia. Rabbits, guinea
pigs and mice were susceptible to the EZ. coli toxin,
However, not every strain of pathogenic coli
elaborated this toxin, nor were all animals sus-
ceptible to it. Of 17 strains of E. coli from various
human infections, only 3 produced this toxin and
only 10% of the animals injected became cata-
tonic. The affected animals recovered within 2%
hr.

1899. Growth characteristics of rickettsiae
in tissue culture. F. M. Bozeman,* H. E.
Hopps,* J. X. Danauskas,* E. B. Jackson
AND J. E. Smaveu. Walter Reed Army Med. Ctr.,
Washington, D. C.

The rickettsiae of epidemic, murine and scrub
typhus, and of spotted and Q fever can be propa-
gated in mouse lymphoblast cells (strain MB III)
grown as a uniform suspension in roller tube
tissue cultures. Using these cells, a system was
developed for quantitative studies on the growth
of Rickettsia tsutsugamushi in proliferating and
colchicine-arrested cells. A known number of cells
was mixed with a given number of infectious units
of rickettsiae, and the course of infection was
followed for 72 hr. by enumeration of cell popula-
tions, determination of mouse LD50’s of cultures,
and microscopic observation of stained smears.
Infected cells in the proliferating cultures in-
creased at the same rate as the noninfected control
cells (doubling in number in 48 hr.), even though
97% of the cells in the infected culture contained
visible rickettsiae 2 hr. after addition of the
rickettsial inoculum. Cultures in which cells were
multiplying or in which multiplication was ar-
rested by colchicine displayed a linear increase in
infectious units of about 3-fold each 24 hr. Pene-
tration of R. tsutsugamushi into MB III cells was

mine tes *- ee a ee

Tolume 15

borate 2
and the
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JACKSON
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March 1956

unaffected by chloramphenicol (20 ug/ml). How-
ever, the ‘antibiotic caused a rapid reduction in
infectious titer of the culture and a rather prompt
disappearance of visible rickettsiae from the cells.
No differences in respiration of infected and non-
infected cells were observed.

1900. Immunization of infants and preschool
children with poliomyelitis vaccine. GORDON
C. Brown AND Dona.p C. Smiru.* Epidemiol-
ogy and Pediatrics Depts., School of Public
Health and Med. School, Univ. of Michigan,
Ann. Arbor.

A study of the antibody response of infants and
preschool children to poliomyelitis vaccine was
conducted. Serological response was measured by
bleeding before and after primary vaccination and
after booster inoculations given 6 months later,
then performing virus neutralization tests in
tissue cultures of HeLa cells. One large group of
children had unintentionally received a poor vac-
cine for their primary stimulus but responded with
good antibody production to a booster inoculation
with a good preparation. Four other groups of
children received potent vaccines on different
primary immunization schedules. In 2 groups re-
ceiving 3 injections of different vaccines at
monthly intervals, the majority of infants as well
as of preschool children responded with increased
neutralizing antibodies to all 3 types of virus.
Another group received 2 injections at an interval
of 1 month. Although most of the subjects re-
sponded well, the postvaccine titers of the infants
were not as high, especially to types I and III. A
final group of infants received only 1 injection to
which they did not respond with demonstrable
neutralizing antibodies. The effect of the booster
inoculation on all groups was marked and even
those not responding well to primary vaccination
showed appreciable development of antibodies.
These included the group of infants who had re-
ceived but 1 dose. Postbooster antibody titers in
these subjects, although not as high as in the other
groups, reflected good response. This study proves
conclusively that vaccination of infants and pre-
school children induces serological responses
comparable to those seen in older age groups.

191. Multiplication of St. Louis encephalitis
(SLE) virus in ascitic tumor cells implanted
in mice irradiated before or after immun-
munization. F. S. CHEEVER AND JAMES F.
Dicxos.* Dept. of Epidemiology and Micro-
biology, Grad. School of Public Health, Univ. of
Pittsburgh, Pittsburgh, Pa.

Adult mice were exposed to sublethal doses of
whole body x-radiation before or after immuniza-
tion against SLE virus. At varying intervals there-
after, these animals and normal controls were
injected intraperitoneally with Ehrlich ascitic

AMERICAN ASSOCIATION OF IMMUNOLOGISTS

583

tumor cells and the ability of these malignant cells
to support the multiplication of SLE virus was
determined. In some experiments virus-infected
tumor cells were injected; in others SLE virus was
injected 8 hr. or 5-6 days after the inoculation of
the tumor cells. The ascitic fluids were harvested
4-6 days after the introduction of SLE virus into
the peritoneal cavities of the mice and titrated for
viral activity. The results may be summarized: /)
ascitic tumor cells implanted in unirradiated im-
munized mice, and in mice irradiated after im-
munization showed little, if any, ability to support
the subsequent multiplication of SLE virus; 2) as-
citic tumor cells implanted in mice irradiated 2
or 3 days prior to immunization supported the
multiplication of virus to a diminished, but
varying, degree as compared to cells implanted in
nonimmunized animals; 3) the injection of virus-
infected ascitic tumor cells (as opposed to unin-
fected cells followed by virus 8 hr. or 5-6 days
later) increased the virus yield in immunized
mice. Lengthening the interval between im-
munization and injection of ascitic tumor cells and
virus decreased it. (Supported by Atomic Energy
Contract no. AT (80-1)-1112).

1902. Infectivity of group A streptococcus.
A. F. Cospurn, H. D. StapE* ann J. M. Nouan.*
Rheumatic Fever Research Inst., Chicago, Ill.
During an outbreak of streptococcal infections

it was noted that type 19, which had been re-

covered sporadically, became responsible for
practically all of the streptococcal upper respira-
tory tract infections. By March 1, the acute
respiratory disease admission rate rose to an
epidemic level and the percentage of throat cul-
tures positive for type 19 hemolytic streptococcus
among men with respiratory symptoms rose from

1 to 45%. Prior to this outbreak, the following

group A organisms were obtained on culturing the

throats of personnel—types 1, 5, 8, 11, 12, 18, 19,

28 and those untypeable. A culture of each organ-

ism was sprayed on 12 mice in air tight chambers.

The streptococcal mortality rate was zero for all

except two types. Type 1 showed 8% mortality and

type 19 showed 58% mortality. At the height of the
epidemic, another type 19 strain (no. 127991) in-
duced streptococcal mortality of 70% in 24 animals
exposed to the aerosol spray. All of these tests were
done on normal Swiss albino mice at room tem-
perature. Pharyngeal tissues of most mice were
infected in warm as well as in cold weather months.

In summer, it was found that 1 wk. after exposure,

all 12 mice challenged with strain N19 had this

organism in the throat flora, but all survived.

Whereas, in winter the majority of mice with

positive throat cultures died of streptococcal

pneumonia with bacteremia. Recent studies show
that, in addition to endogenous infectivity, there

584

is a significant environmental factor which de-
termines whether the carrier state or death is
induced by exposure to aerosol spray.

1903. Action of M5-8450 and helenine against
experimental poliomyelitis in monkeys.
KENNETH W. CocHraNn,* Liana Wer Cuu* AND
Tuomas Francis, JR. Dept. of Epidemiology and
Virus Lab., Univ. of Michigan, Ann Arbor
M5-8450 and helenine are antibiotics derived

from 2 species of Penicillium and which have been

found to protect animals from infection with
certain neurotropic viruses. Although these sub-
stances have not been purified, several investiga-
tors have been impressed with their apparent
similarity, and therefore they are being studied
together. Previous investigations in this labora-
tory have demonstrated the effectiveness of these
substances in monkeys inoculated subcutaneously
with the Mahoney strain of type I poliomyelitis
virus. In view of the importance of the intestinal
tract as a portal of entry and egress for the virus
in poliomyelitis, it was of interest to study the
action of these substances in infections initiated
by orally administered virus. When both M5-8450
and virus were given orally, no protection was
obtained. Six of 6 monkeys given M5-8450 by
stomach tube, at the rate of 100 ml/day for 4 days,
became paralyzed, as did 5 of 6 control monkeys.

That oral infection can be prevented with anti-

biotic is demonstrated by the effectiveness of

helenine given intraperitoneally. Only 1 of 5

monkeys so treated with helenine, 5 ml/kg/day for

4 days, developed paralysis, whereas 5 of 6 control

animals became paralyzed. The incidence of re-

covery of virus from stools of monkeys was re-
duced by helenine treatment. Virus was re-
covered from the stools of surviving control
monkeys for 3 wk., during which time the inci-
dence of virus isolation was 27/32 in the control
animals and 8/31 in the helenine-treated group.

1904. Effects of casein of amino acid mixtures
on nutritional cirrhosis of rats. 8. I. CoHen,
A. J. Patek, Jr., E. Scumatouia, M. Bevans
{introduced by Davip SEEGAL). Columbia Univ.
and Goldwater Memorial Hosp., Columbia (First)
Div., New York City.

Male Sprague-Dawley rats with extensive fatty
and fibrotic changes of the liver were fed for 16 wk.
1) a 30% casein diet, 2) amino acid mixtures (simu-
lating, except for its methionine content, the
amino acid content of the 30% casein diet) with
varying amounts of methionine and/or choline,
or 3) a 4% casein diet. All animals were limited to
equal food intake. Chemical and _ histological
studies were made on collagen and lipid contents
of the livers and the following observations were
based on these. The amino acid mixture containing
0.14% pu-methionine resulted in decreased liver

FEDERATION PROCEEDINGS

Volume 1§

fat, slight increase in body weight, little liver
regeneration and no regression in liver fibrosis,
The amino acid diet containing 1.05% pu-methio-
nine resulted in decrease of liver fat to normal,
moderate growth and liver regeneration, as well as
regression of fibrosis. The amino acid diet with
1.83% methionine resulted in a decrease of liver
fat to normal, moderate growth and regression of
fibrosis, in addition to marked regeneration and
restoration of liver structure. Feeding the diet
composed of the amino acid mixture supplemented
with 0.14% methionine and 0.50% choline resulted
in decrease of liver lipids to normal and some
regression of fibrosis; however, poor growth and
little liver regeneration were noted. The group
fed the 30% casein diet showed decreased liver
fat to normal, marked growth, marked regenera-
tion and restoration of liver architecture, and
marked regression of fibrosis.

1905. Susceptibility of bats to certain en-
cephalitis viruses. Epwin C. CorristTay,
Louts C. La Morte, Jr. aNnD Dorotuy G. Smita
(introduced by Grorce C. Wricut). Camp De-
trick, Frederick, Md.

Several species of bats have been found to be
susceptible to infection with the viruses of both
Venezuelan equine encephalomyelitis (VEE) and
Japanese B encephalitis (JBE). Four species of
bats Eptesicus fuscus, Myotis lucifugus, Pipi-
strellus subflavus, and Corynorhinus rafinesquii
were readily infected with various concentrations
of VEE virus administered either intranasally or
intraperitoneally. In concurrent tests, the sus-
ceptibility of the bats was at least equal to that of
Swiss-Webster albino mice inoculated via either of
the 2 routes. In Eptesicus fuscus and Myotis luci-
fugus, held at room temperature, the blood-virus
concentration reached a relatively high level, at
least 10° mouse i.p. LDs0/ml, within 48 hr. after
inoculation with about 25 mouse i.p. LDso. In some
cases the viremia remained at this level for at
least 26 days. The blood-virus concentration, at
least at specific intervals during the course of
infection, was well above the threshold necessary
to infect some species of mosquitoes. When bats
were infected and stored at hibernating tempera-
ture (10°C) a viremia of low order, 2 to 3 logs, was
maintained for at least 90 days and rapidly rose
when the animals were returned to room tem-
perature. Two species of bats, Eptesicus fuscus
and Pipistrellus subflavus, inoculated subcutane-
ously with JBE virus developed a viremia which
was maintained in some instances for at least 15
days. Present data indicate that neither VEE
nor JBE viruses are lethal for the bats maintained
and tested in this laboratory.

1906. Inhibitors of complement activity. W.
P. CusHMAN* AND E. L. Becker. Div. of Immu-

+ ee ee ee

oe wat® @ = = = 2. @ @ f° @& & it em 6 ee ee ok ue a

—

plume 16

le liver
fibrosis,
methio-
normal,
3 well as
let. with
of liver
ssion of
ion and
the diet
2mented
resulted
id some
wth and
e group
ed liver
»genera-
re, and

iin en-
-RISTAN,
+. SMITH
amp De-

id to be
of both
VE) and
ecies of
}, Pipi-
finesquit
trations
sally or
she sus-
» that of
sither of
tis luci-
od -virus
evel, at
r. after
In some
1 for at
tion, at
purse of
2cessary
ien bats
empera-
ogs, was
dly rose
m tem-
3 fuscus
cutane-
a which
least 15
er VEE

ntained

ity. W.
f Immu-

March 1956

nology, Walter Reed Army Inst. of Research,

Washington, D. C.

Recently Levine (Biochim. & Biophy. Acta, 18:
983, 1955) has shown that either the first (C’1) or
the fourth (C’4) component of guinea pig comple-
ment (C’) is inhibited by di-isopropylfluorophos-
phate (DFP). Independently, Becker (Nature
176: 1073, 1955) demonstrated that it is C’1 which
is inhibited by DFP and proposed that C’l is an
enzyme with esterase activity. If one or more of
the components of C’ is an enzyme, then simple
substances chemically related to the natural
substrate might inhibit the activity of that com-
ponent by competing with the natural substrate.
With this in mind, over 60 compounds have been
tested for their effect on the hemolytic activity of
two 50% units of C’. Simple esters such as ethyl
acetate and ethyl lactate were not inhibitory at
0.05 m. pu-Lysine ethyl ester, pu-Tryptophan
ethyl ester, tosyl arginine methyl ester and pL-
Valine ethyl ester were inhibitory at 0.02 m, while
pL-Tyrosine ethyl ester inhibited at 0.01 m. The
corresponding amino acids or alcohols were not
inhibitory at the same molarities; 0.02 m phenyl-
alanine, 0.02 m tryptamine, 0.005 m cysteine and
0.005 m thioglycolic acid were inhibitory. Acetyl-
dl-tryptophan and acetyl-dl-phenylalanine in-
hibited at 0.02 m, while acetyl-glycine, acetyl-
phenylglycine, acetyl-l-tryptophan and acetyl-d-
phenylalanine inhibited at 0.01 m. Carbobenzoxy-
glutamyl-tyrosine, carbobenzoxy-seryl-tyrosine-
amide and _ carbobenzoxy-glycyl-phenylalanine
were inhibitory at 0.005 m, but carbobenzoxy-
glutamyl-glycine and other nonaromatic amino
acid containing peptides were not inhibitory even
at 0.02 m.

1907. Evaluation of efficacy of influenza virus
vaccine in 1955. FRED M. DAVENPORT AND AL-
BERT V. Hennessy. Univ. of Michigan School of
Public Health, Ann Arbor, Mich.

An influenza virus vaccine containing equal
parts of Swine, PR8, FMI, Conley and Lee strains
of influenza virus, emulsified in mineral oil, was
given to a large group of military recruits training
at Sampson Air Force Base, Geneva, N. Y. The
total concentration of virus in the vaccine was
250 CCA units per dose. A nonvaccinated group of
similar size was maintained as a control. An
outbreak of influenza B occurred early in January,
1955 and the efficacy of the vaccine employed was
measured by comparing the incidence of influenza
in the vaccinated and unvaccinated groups as
determined in hospitalized cases of respiratory
disease by serologic methods. The incidence of
ARD and of infection with hemolytic streptococci
was determined in the same groups of patients by
complement fixation and cultural methods, re-
spectively. It was shown that while the incidence

AMERICAN ASSOCIATION OF IMMUNOLOGISTS

585

of streptococcal disease was approximately the
same in both groups of patients, the incidence of
influenza and of total respiratory disease was
greater in the unvaccinated population. The
amount of protection as measured by serologic
methods and by incidence of total respiratory
illness was in good agreement but less than that
observed in previous studies on the prevention of
influenza B. An explanation for the relatively poor
protection observed was afforded by the observa-
tion that the Lee strains of virus used in the vac-
cine induced low levels of antibody to 1954 and
1955 isolates.

1908. Differential properties in vitro of natu-
ral and immune anti-A and anti-B isoag-
glutinins. IskarL DavipsoHn, Haroip Goop-
MAN* AND Kurt Stern. Dept. of Pathology,
Chicago Medical School, and Mount Sinai Med.
Research Fndn., Chicago, IIl.

An important problem in blood transfusions and
erythroblastosis is the differentiation of immune
from natural anti-A and anti-B agglutinins.
Several criteria presumably permitting such a
differentiation have been described. The present
study is an attempt to gain information on the
reliability and regularity of such differences in
reactivity of antibodies. Blood specimens were col-
lected from approximately 60 healthy volunteers
of group A or B prior to any treatment, and 4-6
wk. after they were injected with either group-
specific substance or with erythrocytes of the
heterologous group. The following tests were done:
titration of anti-A or anti-B agglutinins with
saline as vehicle and diluent, with parallel titra-
tions at 37°C and at 5°C; titrations at 37° C using
papain-treated erythrocytes or erythrocytes
suspended in bovine albumin; titrations following
neutralization with group-specific substance;
hemolysin titration; estimation of avidity. Under
these conditions, a considerable variability in
differential behavior of natural and immune
isoagglutinins became apparent. The following
features were found most regularly and specifically
associated with presence of immune antibodies:
significant isoagglutinin titer when neutralized
serum was tested with papain-treated erythro-
cytes; hemolysin in excess of trace; degree of
avidity. These differential features were inde-
pendent of the titer in saline. No qualitative differ-
ence in behavior of isoagglutinins was noted
depending on whether stimulation was done with
group-specific substance or erythrocytes. (Sup-
ported in part by a research grant (A-77) from the
Natl. Insts. of Health, PHS.)

1909. Nutritional requir ts for propaga-
tion of poliomyelitis virus by HeLa cells in
tissue culture. Harry EaGie anp Karu Ha-
BEL. Section on Exptl. Therapeutics, and Section

586

on Basic Studies, Lab. of Infectious Diseases,

Natl. Microbiological Inst., Natl. Insts. of Health,

Bethesda, Md.

Salts, glucose and glutamine proved to be the
sole components of the medium required for the
propagation of poliomyelitis virus by HeLa cells
in tissue culture. In their absence, the addition of
amino acids, vitamins, serum, purines and pyrim-
idines gave no significant viral response. The
addition of glucose alone, either to such a mixture
or to a simple salt solution usually resulted in a
1-2-log increase in the amount of virus formed;
the addition of glutamine alone resulted in a 2-4-
log increase; while in the presence of salts, glu-
cose and glutamine, the amounts of virus formed
were 3-5 logs greater than those obtained in salt
solution alone. Conversely, on the omission of
glucose from the complete medium, the amount of
virus decreased by approximately 1 log; on the
withdrawal of glutamine, the viral output de-
creased by 3-4 logs; and on the withdrawal of
both glucose and glutamine, the amount of virus
was decreased by 3-5 logs. It would appear that
this cell can make the protein component of polio-
myelitis virus from its own amino acid pool or
proteins. The requirement for glucose and gluta-
mine may be related to the synthesis of viral
nucleic acid.

1910. Gain of two enzymes concerned with
rhamnose utilization by a single muta-
tional event. ELLis ENGLESBERG (introduced
by K. F. Meyer). Biological Lab., Long Island
Biological Assoc., Cold Spring Harbor, N. Y.
Pasturella pestis is unable to utilize rhamnose,

but gives off apparently rare rhamnose-positive

mutants which utilize rhamnose adaptively but
incompletely, accumulating small amounts of
lactic aldehyde. At least 2 enzymes are involved
in the mutation: 1) an 1l-rhamnose isomerase,
which converts 1-rhamnose into a new methyl
pentose, rhamulose (keto rhamnose), and 2) a
kinase, which probably phosphorylates rhamulose
to rhamulose-1-phosphate. There are no traces of
either of these enzymes in the wild type extracts
prepared from cells grown in the medium in which
the R+ mutant readily adapts or in extracts of
unadapted R+.The mutation does not involve the
loss of some ‘soluble’ inhibitor(s), since mixtures
of the R+ adapted extract with the wild type
extract or with the R+ unadapted extract are as
active as the R+ adapted extract alone. Rhamu-
lose was produced enzymatically and separated

from an equilibrium mixture of rhamnose (40%)

and rhamulose (60%) by cellulose column chroma-

tography and fed to the wild type and R+ in a

peptone mineral medium. The R+ mutant adapts

readily to the utilization of rhamulose under these
conditions, while the wild type fails to adapt.

FEDERATION PROCEEDINGS

Volume 16

Furthermore, mutants isolated on media contain-
ing either rhamnose or rhamulose have the ability
to use both these compounds. Thus, it appears that
the wild type lacks the ability (genotype) to pro-
duce both the isomerase and the kinase and that a
single mutation results in a gain in ability to pro-
duce both enzymes.

1911. Measurement of antigen-combining ca-
pacity of antiserums as determined by am-
monium sulfate precipitation of I'* anti-
gen after complex formation with antibody,
Ricuarp 8. Farr. Dept. of Medicine, Univ. of
Chicago, Chicago, Ill.

When I'*!-labeled bovine serum albumin
(I*BSA) is mixed with a 1:10 dilution of normal
rabbit serum in borate buffer, only 3.5% is pre-
cipitated by an equal volume of saturated am-
monium sulfate (SAS). If, however, anti-BSA is
substituted for normal rabbit serum, precipitation
of I*BSA-antibody complexes occurs in the region
of extreme antigen excess where spontaneous
precipitation does not occur in the standard pre-
cipitin test. As in the quantitative precipitin test,
the strength of the bond (avidity) between the
antigen and antibody varies from serum to serum
and must be accounted for in determining the
I*BSA-combining capacity of serum. In contrast
to the precipitin test, many of the difficulties
related to avidity are circumvented because the
SAS-I*BSA method is not dependent upon aggre-
gation secondary to antigen-antibody complexing
and is executed in what otherwise would be the
zone of extreme antigen excess. Anti-BSA serums
are diluted in normal rabbit serum to an arbi-
trarily selected end point where a constant amount
of I*BSA (0.05 y) is precipitated by SAS at 2
different fixed levels of antigen added (0.25 and
0.125 y). The dilutions of antiserums required to
attain these end points are plotted against the
reciprocals of the amounts of antigen remaining
in the supernatant. The antigen combining
capacity of 1 ml of serum at infinite antigen excess
is determined by extrapolation and the slope of
this line is an expression of the avidity of the anti-
body for the antigen. The method ean detect and
characterize nonprecipitating as well as precipi-
tating antibodies.

1912. Detection of nonprecipitating antibody
with radioiodinated antigen. RoBERT FEIN-
BERG (introduced by E. L. Brecker). Walter
Reed Army Inst. of Research, Div. of Immunology,
Washington, D.C.

It was reported (Federation Proc. 13: 1954) that
I'3!_labeled bovine serum albumin (BSA) was not
completely precipitated in the zone of antibody
excess when reacted with certain rabbit anti-BSA
sera, but the label in the supernatant was pre-
cipitable by sheep antirabbit globulin. It was

one fe au Sf SO Mt he

me 15

ntain-
bility
's that
O pro-
that a
0 pro-

1g Ca-
y am-
anti-
body.
niv. of

bumin
ormal
s pre-
d am-
sSA is
tation
region
neous
d pre-
n test,
on the
serum
ig the
ntrast
culties
se the
aggre-
lexing
be the
erums
. arbi-
mount
5 at 2
5 and
red to
st the
aining
bining
excess
ope of
e anti-
ct and
recipi-

ibody
FEIN-
Walter
nology,

1) that
‘as not
tibody
i-BSA
‘s pre-
it was

March 1956

postulatéd that the inability of the labeled antigen
to precipitate in antibody excess was due to
blocking type antibody. The present work is an
attempt to gain evidence as to the nature of this
antibody. Rabbits were injected with human
serum albumin (HuSA) by 3 methods: 1) intra-
venously with alum precipitated antigen, 2) intra-
muscularly with alum precipitated antigen, 3) sub-
cutaneously with antigen plus Freund’s adjuvant.
Serum was collected weekly for up to 26 wk. and
supernatants from antibody excess were tested for
residual labeled antigen. In no bleedings from
rabbits injected by the first 2 methods was labeled
antigen found in antibody excess, whereas in 2 of
the 4 rabbits injected by method three, 13-33% of
the labeled antigen was found in the supernatants
of antibody excess and more than 85% of the lable
was precipitable by antirabbit globulin. Paper
strip electrophoretic analyses, addition of fresh
complement (Maurer. SAB Proceedings, 1955)
dissociation by alkali (STERNBERGER. J. Exper.
Med. 98: 1953) and other techniques were applied
to these sera and their complexes with antigen.
These results and the relationship of this antibody
to other types of nonprecipitating will be dis-
cussed.

1913. Relationship of toxoplasma antibody
activator to properdin system. Harry A.
FELDMAN, Louis PILLEMER AND Louise T.
MiuuER.* State Univ. of New York, Upstate Med.
Ctr. at Syracuse, and the Inst. of Pathology,
Western Reserve Univ., Cleveland, Ohio.
Groonroos has demonstrated that properdin

plus C’2, C’3 and C’4 and Mg** make up the heat
labile accessory factor which was found by Sabin
and Feldman to be necessary for the action of
toxoplasma antibody against that parasite. Our
experiments confirm this report that the properdin
system which is required to activate toxoplasma
antibody is similar to that which is active against
the red cells of patients with paroxysmal nocturnal
hemoglobinuria and which is bactericidal against
gram negative bacteria. The adverse effect of the
properdin system against the gram negative
bacteria and certain viruses occurs in the absence
of antibody, whereas the toxoplasma system is the
first in which the activity of antibody is dependent
upon the presence of properdin.

1914. Antirabies vaccination of man with
HEP Flury virus. JoHn P. Fox, Donatp P.
CoNWELL* AND Puytuis GERHARDT. Dept. of
Tropical Medicine and Public Health, Tulane
Univ. School of Medicine, New Orleans, La.

Since 1953, 257 volunteers have received primary
immunization with living, high embryo passage
(HEP) rabies virus of the chick-embryo-adapted
Flury strain. Immune response was judged by
tests for neutralizing antibodies. The direct

AMERICAN ASSOCIATION OF IMMUNOLOGISTS

587

relation between vaccine dose and response sug-
gested lack of viral multiplication. No clinical
reactions attributable to the virus were observed.
In previous work, undesirably large inocula (2.0
gm of embryo material) were used. Subsequently,
inocula consisted of 0.33 gm intramuscularly or
0.04-0.08 gm intradermally. Apparently best given
3 or 4 times at 5-day intervals, these smaller
inocula elicited rapidly developing immune
response in 85% or more recipients. A similar 4-
dose course of Semple vaccine stimulated antibody
more slowly but in all of 19 volunteers and to
usually higher titers, nearly equal in fact to those
following a full 14-dose course. The rapidity of
response to Flury virus vaccine is gratifying but
more viral antigen may be necessary to insure
100% response. Five to 24 months after primary
immunization with Flury virus, 54 volunteers
received a single booster inoculum. Results indi-
cate that, while HEP Flury virus vaccine ocea-
sionally fails to induce readily demonstrable
primary immune response, it is reliable both in
conditioning the recipient for and eliciting a
booster response. It therefore may be useful for
safely inducing and maintaining antirabies
immunity in high risk groups and for treating
exposed persons with a reliable history of previous
Pasteur treatment.

1915. Propagation of virus in tissue culture
employing a simple maintenance medium
without serum. Murray FRIEDMAN AND MEL-
vIN LIEBERMAN (introduced by Dorotuy
Hamre). Div. of Virology, Naval Med. Research
Unit No. 4, Great Lakes, Ill.

Recent improvements in growth and mainte-
nance media for tissue culture have simplified
virus laboratory operations. However, many
maintenance media contain varying amounts of
different animal sera which may influence the
isolation of new agents since animal sera conceiv-
ably may contain certain viral inhibitors. Further,
the serum in the media stimulates cell growth,
necessitating frequent changing of media to main-
tain an optimal px. A maintenance medium has
been developed which contains no serum and
limits cell growth to a minimum. The medium,
designated LYG, is composed of bacteriological
type media, 0.5% lactalbumin hydrolysate, 0.1%
yeast extract, 0.5% gelatin in Hanks balanced
salt solution. This medium has been compared
with other maintenance media, 199 with and
without 5% horse serum for the growth of influ-
enza B, Polio I, Coxsackie (Conn-5) and APC type
4 viruses in cultures of HeLa cells and/or monkey
kidney. With the exception of Coxsackie virus in
HeLa cells, these viruses grew equally well with
the new medium when compared to the other
media. Growth of Coxsackie virus was demon-

588

strated in monkey kidney but not in HeLa cells
using LYG medium. Titrations of the viruses
produced in the different media revealed no
differences in the total virus growth with any of
the media tested with one tissue culture system.
This economical and simple LYG medium is now
being employed for the isolation of respiratory
disease viruses and for the production of APC
antigens.

1916. Experimental typhoid fever in chim-
panzees: II. Prophylactic immunization.
Stipney Garnes,* Maurice LANpy, GEOFFREY
EpsaLtt AND RosBert TrapAani.* Walter Reed
Army Inst. of Research, Washington, D. C.

Small groups of chimpanzees were inoculated
with the agents noted below, and challenged orally
with approximately 5 thousand million organisms
from a 6-hr. culture of S. typhosa, strain Ty2,
carried through either 2 or 3 chimpanzees prior to
use. Except as otherwise noted, vaccines or anti-
gens were given subcutaneously at 0, 2, 4 and +12
wk., with the challenge about 2 wk. later. Vaccines
were given in 0.5 cc doses, antigens in 0.04 mg
doses. The course of infection was followed by
daily temperatures, stool cultures and blood cul-
tures. Results were as follows: acetone-killed and
dried vaccine, prepared from strain Ty2, fully
protected 10 animals; 5 of 7 controls manifested
frank infection, 2 showing unapparent infection.
Vi antigen fully protected 2 of 8 (although infec-
tion in others of this group was minimal); O anti-
gen protected none of 4, and 4 of 5 controls were
infected. In a later experiment heat-phenol vac-
cine (S. typhosa strain 58) protected 4 of 5 animals.
When the interval from 4th (booster) dose of
antigen to challenge was lengthened to 12 wk.,
acetone-killed vaccine protected 2, or possibly 3,
of 4 animals. It is concluded that both types of
vaccine used provided significant protection in
chimpanzees against the challenge employed, but
that the efficacy of the purified antigens as used in
these studies was less clearly established.

1917. Persistence and fate of S**-labeled anti-
gen in livers of normal and immunized rab-
bits. Justine S. Garvey* anp Dan H. Camp-
BELL. California Inst. of Technology, Pasadena.
Investigation of the persistence of S*5-labeled

hemocyanin has been continued (J. Immunol. 72:

131, 1954, and zbid., in press). Altered, but non-

dialyzable antigen fragments have now been de-

tected as long as 20 wk. after a single injection or
after the last of several injections. The initial loss
is more rapid in immunized animals, but even-
tually levels off in all animals in a few weeks to
about 1% of the amount injected. About 75 % of
the tagged material can be extracted from liver
tissue with sucrose solution. By adsorption of the
dialyzed sucrose extract on Dowex-2 resin and

FEDERATION PROCEEDINGS

Volume 1§

subsequent elution with sodium salicylate,
product is obtained which contains most of the
radioactivity and only 1% of the protein which was
in the solution before adsorption. The eluate con-
tains, principally, 2 electrophoretic components
(1 = 7.3 and 12.0). These components are both
radioactive and can be separated by salt precipita-
tion. The fast component is associated with ribo-
nucleic acid and the total amount, as well as
specific radioactivity, decreases with time. The
ability of material to precipitate antibody de-
creases with time but specific inhibition as well as
specific coprecipitation can be detected at least
8 wk. after injection. Antigenicity and molecular
size of the antigen fragments are being investi-
gated at the present time.

1918. Reactivity of various media to produc.
tion of staphylokinase. Earu B. GEeruHem
AND Howarp R. Maxwe .u.* Pharmacology
Dept., Sherman Labs., Detroit, Mich.

A previous report (Federation Proc. 12: 444,
1953) indicated that staphylokinase and the
bacterial factor concerned with coagulase could
be quantitatively separated from 48-hr. cultures
of Staph. aureus grown in brain heart infusion.
This was preferentially carried out with absolute
methanol fractionation and the adjustment of the
pH. The previous work has been extended by grow-
ing Staph. aureus strains in or on the following
media: 1) brain-heart infusion, 2) brain-heart
infusion agar, 3) Difco no. 110 agar and 4) simu-
lated no. 110 liquid medium. This last medium
lacked agar and amino acids.were added to approx-
imate the gelatin. The precipitation techniques
involved a comparison with methanol, ethanol,
specially denatured ethanol, and solox; these
agents were used with and without pH adjustment.
This study is primarily concerned with maximal
yields of staphylokinase.

1919. Effect of pH on metabolism of strain
HeLa cells and on replication of poliovirus.
GeorcE E. Girrorp,* Hucu E. Rosertson*
AND JEROME T. SyverTOoN. Dept. of Bacteriology
and Immunology, Univ. of Minnesota, Minng-
apolis.

HeLa cells in culture have been used widely for
theoretical and practical studies of poliovirus
infection. Since it has been shown that chemical
factors affecting virus production affect host cells,
physical factors influencing cells may also alter
virus yield. The present study is concerned with
the effects of culture medium pu. Media varying
in pH from 6.3 to 8.5 by increments of 0.2 were
prepared with 0.01 m phosphite, 0.01 m phosphate
and 0.005 m tris (hydroxymethyl) aminomethane
as supplementary buffers. These buffers are not
toxic for HeLa cells. The growth medium on dupli-
cate bottle cultures of strain HeLa was replaced

ste

wind

plume 15

‘late, 4
t of the
1ich was
ate con-
ponents
re both
ecipita-

th ribo. |

well as
ne. The
»dy de-
| well as
at least
plecular
investi-

roduc.
ERHEIM
vacology

2: 444,
nd the
e could
ultures
fusion.
bsolute
t of the
y grow-
llowing
n-heart
) simu-
nedium
upprox-
uniques
thanol,
; these
stment,
1aximal

strain
ovirus.
RTSON*
orvology
Minng-

lely for
iovirus
1emical
st cells,
o alter
-d with
yarying
2 were
»sphate
ethane
are nob
1 dupli-
»placed

March 1956

with the media of varying pu, the cells were in-
fected commonly with 25 TCID of poliovirus, and
the cultures were incubated at 36°C until cellular
destruction was observed. During incubation, the
medium was partially replaced at 6-12-hr. inter-
yals. At pH 6.7 or less, virus production was re-
tarded significantly or reduced. The effect of virus
production was shown not related to decreased

| stability of released virus at lower px levels.

Decreases in host cell oxygen consumption and
glucose utilization were correlated with decreased
virus yield at lower pu.

1920. Masked viral infection of HeLa cell cul-
tures. Harotp S. GINSBERG AND GEORGIANA
§. Borer.* Dept. of Preventive Medicine, West-
ern Reserve Univ. School of Medicine, Cleveland,
Ohio.

The emergence of cytopathogenic viruses from
tissue cultures of tonsils and adenoids suggested
that these agents persist in man as latent infec-
tions. Studies of the biologic characteristics and
epidemiologic behavior of these viruses (variously
called AD, APC, RI, or AD-ARD group) lent sup-
port to this hypothesis and suggested that it
might be possible to establish a model of this
latent infection in tissue culture. Type 3 and 4
viruses were employed. HeLa cell cultures were
infected with 104-° TCDso of virus, following which
the cells were passaged in series using as growth
medium 40% human serum and 60% Hanks’
balanced salt solution. Although these serial
cultures contained virus titers from 107!-° to
10-?-5, the HeLa cell cultures appeared uninfected.
When growth medium was removed, cells washed,
and maintenance solution added, however, typical
cytopathic alterations occurred. Human serum,
unless specially selected, contained type-specific
antibodies for the viruses employed. Infected
cultures have been initiated and passaged in series,
however, using human serum in which neutralizing
antibodies were not detectable. Moreover, when
rabbit specific antiserum was incorporated in the
maintenance solution, cytopathogenic effects were
not prevented. Thus, although it has not been
possible to eliminate antibody as an important
factor for maintenance of the infection in a
‘masked’ state, it appears that conditions for
active cell growth are essential. It cannot be
implied from these data that this is a model of a
latent viral infection of man, but these studies do
suggest a mechanism by which persistent viral
infections can occur.

1921. Attenuation by histamine of lethal ir-
radiation effect in mice and its potentia-
tion by Benadryl. Horace Gotprz, Ganson J.
TARLETON,* JosEPH G. GorDON* AND Gus-
TtavEous L. GrercrrR.* Lab. for Exptl. Oncology

eerrre

AMERICAN ASSOCIATION OF IMMUNOLOGISTS

589

and Dept. of Radiology, Meharry Med. College,

Nashville, Tenn.

Doses of Benadryl well tolerated by normal
mice (1 mg) reduced considerably the survival
span of mice irradiated with lethal doses of fil-
tered x-rays: vice versa, the Histamine (10 mg)
extended (indefinitely in 22.5% of animals) the
survival of animals treated by the same technique
and with the same amount of irradiation. An irra-
diation dose of 600 r was lethal for all mice with
only a narrow range of variation in their survival
span. Mice of CAF! strain of about 25-gm weight
were used throughout. The effect of each drug
decreased with the increase of the interval be-
tween pretreatment and irradiation. The injection
after treatment (within 5-10 min. following
irradiation) had an effect similar to pretreatment
but with a wide range of variations. These data
suggest an interpretation that a short-lasting,
presumably vascular reaction induced in the ani-
mal by irradiation was potentiated by Benadryl
and inhibited by Histamine. The partial neutrali-
zation effect of Histamine by subsequent Benadryl
administration, and to a lesser extent vice versa,
may be considered as an evidence for the spec-
ificity of their action in irradiated mice. More-
over, the lack of complete neutralization suggests
that a major part of each compound became,
immediately after injection, inaccessible for its
antagonist, possibly by irreversible fixation on
tissues. With regard to study of these compounds
in other animal species, it should not be forgotten
that the maximum tolerated dose of Histamine is
relatively high for the mouse and low for humans.
It may be noted also that the reported therapeutic
effect of Benadryl in radiation sickness is not in
contradiction with our data, since radiation sick-
ness and lethal effect of total body irradiation do
not imply identical body reactions. The results
briefly reported above may outline a new approach
to the study of lethal effect attenuation in irradi-
ated animals. (Supported by Atomic Energy Com-
mission Grant AT-(40-1)-1993).

1922. Ascites tumors in experimental animals
and peritoneal carcinosis in humans. Hor-
ACE Gotp1zE, MatrHEw WALKER* AND LOUISE
Keutey.* Lab. for Exptl. Oncology and Dept. of
Surgery, Meharry Med. College, Nashville, Tenn.
There is a tendency in oncology to consider

‘ascites tumors’ in mice and rats as an artificial

condition, induced only by experimental transfers,

and therefore essentially different from sponta-
neous malignant ascites in humans; it is attempted
to support this sharp separation by reference
to the abundance of free tumor cells in experi-
mental ascitic fluid as compared with their scar-
city in human ascites. However, cases of spon-
taneous ascites in mice occur not infrequently

590

following the spread of subcutaneous or intra-
muscular tumor growth from the abdominal wall
or the leg into the peritoneal cavity. High tumor
cell concentration in ascitic fluid is usually at-
tained only after several serial i.p. transfers.
Thus, the human condition is comparable to
ascites tumors before growth potentiation of their
cells by serial transfers. Both in animal and human
conditions, production of ascites follows implanta-
tion into the peritoneal serosa of tumor cells
either from ingrowing tumor tissue (in spontane-
ous ascites) or from inoculated tumor tissue sus-
pension (experimental ascites). In mice this pri-
mary implantation of tumor cells (before ascites
formation) can be easily distinguished from the
secondary implantation of free tumor cells pro-
liferated in the ascitic fluid. Using a method of
transuterine implantation of tumor cells into the
serosa covering female genital organs, we were
able to induce ascites with any available mouse
tumor strain. This corresponds to the clinical ob-
servation about frequent association of ascites
with tumor implants in the Douglas space. Mas-
sive implantation of tumor tissue fragments or
cell clumps into the peritoneal serosa of the
mouse resulted in the growth of numerous easily
bleeding tumor nodules (bloody fluid) closely
similar to human miliary carcinosis with bloody
ascitic fluid reported by Ribbert. Some authors
described organized tumor cell implants as ‘‘solid
phase of ascites tumors’”’ and tumor cell infiltra-
tion of connective and fat tissue as ‘‘semi-solid
tumors.”’ ‘“‘Ascitic type of tumor growth’’ is
another term for peritoneal localization of tumor
growth. This substitution of new terms for terms
established in pathology is unnecessary, since
“ascites tumor”’ is a pathological condition of the
peritoneal cavity essentially similar (with varia-
tions due to difference in species, size of the body,
its vertical position in humans, etc.) to peritoneal
carcinosis in humans. (Grants from American
Cancer Society and Natl. Cancer Inst., Natl.
Insts. of Health, PHS.)

1923. Uptake of transforming DNA and its
penefration into Hemophilus influenzae.
Sot H. GoopGat AND RoGer M. HeErrriorrt (in-
troduced by B. F. Cuow). Dept. of Biochemistry,
Johns Hopkins School of Hygiene and Public
Health, Baltimore, Md.

The uptake of P*-labeled transforming DNA
by susceptible Hemophilus influenzae follows 2
different patterns. Some of the adsorbed DNA and
TP is resistant to DNAse or washing and the quan-
tity is directly related to the number of cells trans-
formed. The remainder of the DNA and TP can be
washed off or digested by DNAse and the quantity
does not appear to be related to the number of
cells transformed. In the latter case the eluted
DNA and TP are equivalent to a fraction of the

FEDERATION PROCEEDINGS

Volume 1§

original TP solution, i.e., there was no fractiona-
tion or destruction by the uptake and elution,
Shortly after adsorption, but before phenotypic
expression has taken place, the TP which is resist-
ant to DNAse can be detected in disrupted cells,
This TP is now DNAse sensitive and TCA pre-
cipitable. Assuming a molecular weight for DNA
of 6,000,000 the following relationships may be
noted: 1) a single cell of Hemophilus influenzae
contains approximately 300 molecules of DNA;
2) the uptake of DNAse resistant DNA by the
cell population involves 350 molecules per trans-
formation; 3) the maximum frequency of trans-
formation in our hands is one per 350 cells. (This
work was supplemented by a grant from the
Atomic Energy Commission.)

1924. Use of chicken antisera for rapid deter-
mination of plasma protein components.
Morris GoopMaNn, Davin S. Ramsey,* WiL-
LIAM L. Simpson,* Dona.Lp G. Remp,* Dante.
H. Basinski* AND MicHaEL J. BRENNAN,*
Detroit Inst. of Cancer Research and Henry Ford
Hospital, Detroit, Mich.

Routine clinical use of the immunochemical
method for quantitating plasma proteins has been
largely prevented by the difficulty encountered in
systematically producing specific antisera of
satisfactory potency. Our study demonstrates that
this difficulty can be overcome by the use of chick-
ens. With Cohn fractions as the source of the
antigens, satisfactory antisera were readily pro-
duced to human albumin, gamma globulin, fibrin-
ogen and orosomucoid. By employing only one
injection per bird, antibody production to con-
taminants was minimal, and each antiserum was
found by Oudin tests to be specific for a single
antigenic species. Identical antigen assays were
obtained with antisera before and after partial
absorptions with heterologous Cohn fractions.
Reactions were carried out in 8% NaCl and were
measured by a rapid nephelometric technic. For
each protein being analyzed a single dilution of
human serum could be chosen which placed the
reaction of the abnormal, as well as the normal
samples, on the calibration curve obtained with
known amounts of the protein. The values for
gamma globulin and albumin determined immuno-
chemically on a large number of sera for normal
adults and patients corresponded well with com-
panion values obtained by paper electrophoresis
and with the cited values for normals established
by moving boundary electrophoresis. Anomalous
results with certain multiple myeloma sera empha-
sized the theoretical importance of quantitating
plasma proteins on the basis of antigenic speci-
ficities.

1925. Cross reactions between antisera to dif-
ferent human cell strains. KARL HABEL AND

ot!

19:

pe ae ee

E XUM

olume 16

actiong-
elution,
snotypic
is resist-
ed cells,
CA pre-
or DNA
may be
fluenzae
f DNA;
_ by the
r trans-
f trans-
ls. (This
‘om the

i deter-
onents,
* Wits
DANIEL
ENNAN,*
ary Ford

hemical
1as been
tered in
sera of
ites that
of chick-
» of the
ily pro-
1, fibrin-
nly one
to con-
‘um was
a single
ys were
partial
actions.
nd were
nic. For
ution of
iced the
normal
ed with
lues for
mmuno-
normal
th com-
phoresis
ablished
omalous
empha-
titating
¢ speci-

to dif-
BEL AND

March 1956

Bethesda, Md.

Antisera were produced in chickens and rabbits
with cell suspensions of HeLa, KB and normal
human skin cells grown in horse serum-fortified
medium in tissue culture. Antigens were prepared
from these same cell lines grown in human serum-
fortified medium. When tested for cytotoxic
effect on cells grown in a monolayer sheet on glass,
for agglutination of suspended cells and for com-
plement fixation, cross reactions were demon-
strated between all the antisera with each of the
antigens. Chicken antisera were found to be
superior to rabbit antisera in the agglutination
test, but rabbit antisera were more cytotoxic and
had good complement-fixing antibody titers.

1926. Amino acid requirements of rabbit fi-
broblasts, strain RM3. R. F. Harr* anv H. E.
Swim* (introduced by R. F. Parker). Dept. of
Microbiology, Western Reserve Univ. School of
Medicine, Cleveland, Ohio.

A strain of fibroblasts, designated as RM8,
derived from adult rabbit muscle has been serially
propagated in vitro for 18 months. Strain RM3 is
highly susceptible to infection with the CVII
strain of vaccinia virus and multiplication of the
virus is accompanied by complete destruction of
cells. In anticipation of studies concerning factors
affecting cellular susceptibility and virus multi-
plication, the nutritional requirements of these
cells have been investigated with particular refer-
ence to their amino acid requirements. The experi-
mental media were composed of various chemically
defined mixtures supplemented with 2% (V/V)
dialyzed horse serum. Cellular proliferation ceased
when each of the following compounds was omitted
from the medium: arginine, cystine, glutamine,
histidine, isoleucine, leucine, lysine, methionine,
phenylalanine, serine, threonine, tryptophan,
tyrosine and valine. The concentration of each of
the 13 amino acids and glutamine required for
optimal growth under conditions of the experi-
ments has been established. The amino acids fall
into 2 distinct groups with respect to the cellular
response to a single omission. When cystine or
glutamine is removed from the medium the cells
degenerate rapidly, whereas no distinct cyto-
logical changes are observed when any of the
remaining 12 amino acids are omitted. This lack of
cellular response to amino acid deficiencies is in
direct contrast with the results obtained with
other strains of cells. The potential significance of
these observations will be discussed in addition to
the quantitative aspects of these studies.

1927. Serological survey for certain mosquito-
borne viral infections in Luzon, Philippine
Islands. W. McD. Hammon, G. E. SatHer,*
W. D. Scurack, Jr.* anp B. J. Mruier.* Dept.

AMERICAN ASSOCIATION OF IMMUNOLOGISTS

591

of Epidemiology and Microbiology, Grad. School

of Public Health, Univ. of Pittsburgh, Pitts-

burgh, Pa.

A serological survey has been made for anti-
bodies to certain mosquito-borne viruses in the
native population of part of Luzon. Fifteen
viruses, Japanese B, Murray Valley, West Nile,
St. Louis, eastern and western equine, Ilheus,
Zika, Ntaya, Semliki Forest, Bunyamwera,
Bwamba, Uganda, and dengue types I and II were
included. Neutralization tests were employed for
all and in addition hemagglutination-inhibition
(HI) was used for dengue. All sera were collected
in 1953, and represented 4 native population
groups, principally children and including Ne-
gritos. When a sufficient quantity of serum was
available, tests were made with all viruses; other-
wise Japanese B, at least, was used. A total of 249
sera was tested. Sera was found in all groups
which neutralized one or more of the following
viruses: Japanese B, Murray Valley, West Nile,
St. Louis, Ilheus, Zika, Ntaya, Semliki Forest and
dengue types I and II. Many negative to Japanese
B were positive to one or more of the other agents.
No sera neutralized eastern or western equine,
Bunyamwera, Bwamba or Uganda viruses. Evi-
dence of dengue infection, particularly in the
very young, was surprising in view of the reported
absence of clinically diagnosed dengue for a num-
ber of years. Cases of encephalitis resembling
those due to the arthropod-borne group of viruses
are not uncommon in the area, and in addition
there are many fevers of unknown origin. Since
many of the viruses under study are antigenically
related serological studies do not determine
exactly how many or which viruses are present.

1928. Characteristics of a series of antigenic
variants of influenza A virus. DoRrotTuy
HamMrRE, Paut GERBER* AND CLAYTON G.
Loostt.* Section of Preventive Medicine, Univ. of
Chicago, Chicago, Ill.

A primary series of 5 antigenic variants of influ-
enza A virus (PR8 strain) has been developed by
passage in partially immunized mice. With this
procedure a second series was initiated by passage
of the third variant in mice immunized with parent
PRS8 virus. The antigenic characteristics of these
variants were then studied employing cross
hemagglutinin-inhibition tests, cross neutraliza-
tion tests in ovo and antibody absorption experi-
ments using convalescent ferret antisera. These
variant strains and their parent PR8 virus were
also compared with respect to antigenic potency
and mouse pathogenicity. Cross hemagglutination-
inhibition tests with other known influenza A
viruses were also carried out. The results of these
tests showed a decrease in PR8 component in the
antigenic composition of the variants. This was
accompanied by an increase in at least 2 new com-

592

ponents whose antigenic potency was less than
that of PR8 although no change in mouse patho-
genicity could be detected. Although marked
antigenic changes could be demonstrated among
the induced strains, these variants still showed
sufficient relationship to the original PR8 strain to
place them in the A group rather than in the A
prime or swine group of influenza viruses.

1929. Transfer of lymph node cells incubated
in vitro with filtrates of trypsin-treated
suspensions of Shigella paradysenteriae.
T. N. Harris, Susanna Harris,* Cuirron A.
OapuRN* AND Miriam B. FarBer.* Children’s
Hosp. of Philadelphia and Dept. of Pediatrics,
Univ. of Pennsylvania, Philadelphia.

In a previous communication it was reported
that lymph node cells from rabbits which had not
been injected with Shigella paradysenteriae were
incubated with rabbit serum which had been
treated with these organisms. After transfer of
such cells to fresh, irradiated recipient rabbits,
agglutinins to the Shigella appeared subsequently
in the sera of these animals. These and other
data suggested that antigenic material of the
immunologic reactivity of the Shigella agglutin-
ogen was present in solution in the Shigella-
treated serum, in a form effective for cell-transfer
experiments. In more recent experiments it has
been found that if suspensions of Shigella are
treated with trypsin and then cleared of the
bacterial cells by Seitz filtration, the filtrates are
effective in lymph node cell transfer experiments
in the same way. Control suspensions, incubated
without trypsin, also appear to have, in solution,
material similarly effective in cell-transfer. In
such experiments, and by serologic measurements,
the filtrates of trypsin-treated suspensions give
evidence of having substantially higher concentra-
tions of the antigenic material than do filtrates
of control suspensions. Preparations of filtrates
of trypsin-treated suspensions of Shigella which
had been dialyzed retained the antigenic material.
In addition, equilibrium dialyses (Visking casing)
of such filtrates were carried out and the dialysates
thus prepared were also found t contain material
effective in cell-transfer. The active material in
the dialysate is retained by a cellophane mem-
brane which allows free dialysis of small peptides,
and is precipitable by 3 volumes of ethyl alcohol.

1930. Applicability of ‘Doctrine of Original
Antigenic Sin’ to influenza B by determin-
ing antibody response after vaccination
with monovalent type B influenza virus
vaccines. ALBERT V. HENNESSY AND FRED M.
Davenport. Univ. of Michigan School of Public
Health, Ann Arbor, Mich.

Previous studies on the distribution by age of
antibody to Lee (1940) Allen (1945) and Longhway

FEDERATION PROCEEDINGS

Volume 1§

(1952) strains of type B influenza virus, when
correlated with the known periods of prevalence
of influenza B, indicated that as with influenza A,
the initial infections of childhood orient the anti.
body response to subsequent experience with
antigenic variants of type B influenza virus. To
obtain further information about the antigenic
experience of 3 segments of the population,
children, aged 4 to 11 yr., military recruits, and
persons over 30 yr. of age were vaccinated with
monovalent vaccine containing either Lee, Allen,
Longhway or Great Lakes (1954) strains and their
antibody response was measured with these
viruses and a strain isolated in 1955. Children
born after the period of prevalence of the Lee
strain did not develop significant levels of anti-
body to Lee virus unless that strain was given by
vaccination. However, regardless of the vaccine
used, children did develop marked increase in
antibody to strains prevalent during the period of
their initial infections or to strains closely related
antigenically. In contrast, antibody levels to Lee
virus in recruits were reinforced by all of the
vaccines given. These individuals were children
at the time Lee virus was isolated. The richness of
the antigenic exposure of persons over 30 was
reflected by diminished antibody response to all
strains included in the vaccines. Thus, data ob-
tained support the thesis presented.

1931. Action of Clostridium tertium enzymes
on blood group substances. CALDERON Howe,
Joun D. MacLEnNAN* AND Exvin A. Kasat.
College of Physicians and Surgeons, Columbia
Univ., and Neurological Inst., New York City.
Among 30 typical strains of Clostridium tertium,

19 produced enzymes specifically splitting blood

group A substance. Growth requirements of one

strain (Iseki) and optimal conditions for enzyme
production were determined. Enzyme was esti-
mated under standard conditions by capacity to
inactivate serologically the A antigen in purified
fractions of hog mucin and individual hog stomach
linings, residual blood group activity being meas-
ured by hemagglutination-inhibition and quanti-
tatively by precipitation with calibrated anti-A
sera. Enzymic activity was present in dialyzed
lyophilized supernatants of cultures grown in 4
semi-defined medium containing glucosamine, and
was inhibited by 10-* m Ni**, Zn** and Cut", but
not by Ca++, Mn++, Cot++, Mgt or Fe*. Activity
was optimal between px 7.2 and 7.8 against blood
group A antigen, both in soluble form and intact
erythrocytes, and against M and N antigens; but
no effect was observed on B, O(H), Rh Kell or

Duffy antigens. Receptors for Columbia SK virus

hemagglutinin were destroyed; those for influenza

A and B were unaffected. All erythrocytes, regard-

less of blood group, were rendered panagglutin-

oh = 8° mm se we OeleelU Ml COtlUetlCUe

—

= => <

io: fn - ee

a Qa eos 8

olume 16

8s, when
evalence
lenza A,
he anti-
ce with
irus. To
ntigenic
ulation,
lits, and
ted with
e, Allen,
nd their

vaccine
rease in
yeriod of
y related
s to Lee
1 of the
children
shness of
30 was
se to all
data ob-

nzymes
1 Hows,
KaBat,
Yolumbia
ork City.
_ tertium,
1g blood
s of one
enzyme
vas esti-
acity to
purified
stomach
ng meas-
| quanti-
d anti-A
dialyzed
wn in 4
\ine, and
ut, but
Activity
st, blood
id intact
ens; but
Kell or
SK virus
influenza
, regard-
1gglutin-

March 1956

able. Peptidase activity against selected di- and
tripeptides was also demonstrable. Enzyme
treatment of purified blood group substance from
hog mucin (O(H) + A) completely inactivated
only the A antigen, and liberated dialyzable hexos-
amine, 80% of which was N-acetyl hexosamine.
One enzyme fraction, obtained from culture super-
natant by precipitation with ammonium sulfate
between 42 and 52% of saturation, contained a
single electrophoretic component, and completely
inactivated purified hog mucin A antigen within
1hr.

19382. Differentiation of poliomyelitis, Cox-
sackie and ECHO (enteric cytopathogenic
human orphan) viruses by plaque morphol-
ogy and host-cell susceptibility. G. D.
Hsrune* anp JoserpH L. MELNicK. Section of
Preventive Medicine, Yale Univ., New Haven,
Conn.

Monkey kidney cells were found to survive
under agar for much longer periods in stoppered
flat bottles than in unsealed Petri dishes kept in a
humidified CO2-air mixture. This enabled plaque
formation to occur with Coxsackie and ECHO
viruses which have a delayed cytopathogenicity,
often requiring 5-12 days to become visible, as
against the 2-4 days required for poliovirus.
Plaque morphology of the ECHO viruses studied
(types 1, 3, 4, 6, 7, 8, 9), and of other antigenically
distinct orphan viruses not yet classified by the
Committee on ECHO Viruses of the National
Foundation for Infantile Paralysis, was suffi-
ciently distinctive, except for type 7, to permit
their differentiation from poliovirus plaques.
The plaques of the ECHO viruses had irregular
diffuse boundaries, and healthy cells were found
within the degenerated areas. Plaques of Cox-
sackie A9 virus and those of the rapidly appearing
polioviruses were indistinguishable. The group B
Coxsackie viruses produced circular plaques but
with diffuse boundaries and fluffy centers. The
enteric viruses studied could also be differentiated
by their host range, for kidney cells of different
monkey and baboon species vary in their suscepti-
bility to these viruses. For example, rhesus (Ma-
caca mulatta) and cynomolgus (M. irus) were
susceptible to all viruses studied. Cells from the
African red grass, military monkey (Erythrocebus
patas) were found to be 2-3 times more susceptible
than those from rhesus to poliovirus and to the
group B Coxsackie viruses, but they proved re-
sistant to Coxsackie AQ virus and to all the ECHO
virus types studied except type 7.

1933. Effect of cortisone and X-ray on periph-
erally induced Japanese B_ encephalitis
virus infection in hamsters. Imam Z. E.
ImMam* anp W. McD. Hammon. Dept. of Epi-
demiology and Microbiology, Grad. School of

AMERICAN ASSOCIATION OF IMMUNOLOGISTS

593

Public Health, Univ. of Pittsburgh, Pittsburgh,

Pa.

Approximately 90% of Syrian hamsters inocu-
lated subcutaneously with 10% mouse brain sus-
pensions of Japanese B encephalitis (JBE) virus
usually remain free from visible signs of infection.
Viremia appears within 6 hr. and disappears within
4 days. Virus appears in the brain on the 4th day,
reaches a maximum titer (10-*-*) on the 7th and
8th days and disappears by the 14th day. Serum
neutralizing antibodies begin to appear by the 5th
day. Those few hamsters showing illness attain
brain titers of about 10~5-5. In order to render all
animals susceptible to disease from peripheral
injection of high dilutions of JBE virus the effects
of varying doses of cortisone and x-ray were
tested. Cortisone alone or lower doses with x-ray
were effective in tolerated dosages. Cortisone
alone, 0.2 mg/gm b. wt., intramuscularly, was
finally selected for the definitive work. In such
hamsters the LDso dose of JBE virus was approxi-
mately 10-* when given subcutaneously in a 0.1
ml dose. Virus titers of the brain of sick animals
were not infrequently in excess of 10°. This
method was thus found satisfactory for rendering
animals highly susceptible to peripheral inocula-
tion. It is believed that the mechanism involved
is the retardation of antibody formation, per-
mitting unlimited multiplication of virus in the
brain. A preliminary experiment using cortisone-
treated hamsters inoculated with St. Louis virus
has given 80% fatality from subcutaneous inocula-
tion of a 10% mouse brain virus suspension.

1934. Properties of early host-virus complex.
Nakao IsHrpa* AND W. WILBUR ACKERMANN.
Dept. of Epidemiology and Virus Lab., Univ. of
Michigan School of Public Health, Ann Arbor.
The extent of the initial reaction between virus

and cell which forms a complex is determined by

the concentration of reactants and the time of
exposure. Thus, after any interval, the reaction
may be terminated by separating the reactants.

By estimating the amount of complex formed in

that interval, the rate of reaction can be deter-

mined. When the virus is influenza and the host
cells the chorioallantoic membrane, the cells
are easily removed from the virus. The remaining,
susceptible, uninfected cells can be inactivated
with receptor-destroying enzyme and the number
of infected cells can be estimated in terms of
their capacity upon prolonged incubation to
produce virus. The reaction when followed in
this manner was found to proceed rapidly both at
3° and 37°C. Accompanying the reaction is a loss
of detectable viral infectivity; however, the
host-virus complex retains its susceptibility to
the inhibitory action of specific immune serum.
This complex, while susceptible to the effects of

594

serum, appears resistant to the action of receptor-
destroying enzyme. Upon incubation at 37°C,
the initial complex undergoes a further reaction
which is characterized by a loss of sensitivity to
immune serum. This second temperature-depend-
ent reaction proceeds more slowly than the initial
reaction and its rate is determined in part by the
concentration of virus used to produce the original
complex. Thus, the 2 reactions can be distin-
guished by their different rates, their temperature
dependence and the _ susceptibility of their
products to the inhibitory action of immune
serum.

1935. Immunization against smallpox. E. B.
Jackson, B. L. ELisBere* anp J. E. SMADEL.
Walter Reed Army Med. Ctr., Washington, D. C.
Jennerian prophylaxis had undergone few

changes during the 150 yr. of its successful em-
ployment. Neverthless, calf lymph and the per-
cutaneous route of inoculation have certain dis-
advantages, particularly for mass immunization.
We, like others before us, increased stability of
vaccine virus by lyophilizing bacteriologically
sterile suspensions of infected chorioallantoic
membranes (CAM) of hens eggs. Dried CAM vac-
cine retained the desirable properties of calf
lymph and remained highly infectious when
stored for years at 4°C or for months at 37°C. The
jet injection principle was employed for adminis-
tering 0.5 ml of CAM vaccine, containing approxi-
mately 100,000 infectious units of virus, to non-
immune infants and to soldiers whose immunity
varied. The jet deposited a small portion of
the inoculum intradermally and the remainder
subcutaneously. The cutaneous reactions elicited
by jet injection of CAM vaccine were essentially
indistinguishable from those obtained with calf
lymph vaccine administered by multiple pressure;
moreover, when both procedures were applied
simultaneously to the same person the results
were comparable. Nonimmune infants given CAM
material by jet injection developed hemagglutinin
inhibiting (HAI) antibodies titering 1/128-1/1024.
Among 1000 recruits vaccinated by this procedure
9% displayed primary, 53% accelerated and 38%
immediate responses. Only 1 man failed to show
1 of the 3 classical reactions. HAI antibodies
appeared or increased in more than half the
vaccinated recruits selected for serologic study
and maintained levels of 1/16-1/256 in the re-
mainder; postvaccination levels were similar to
those of the control group immunized percutane-
ously with calf lymph. Jet injection of CAM vac-
cine is a rapid, simple and effective procedure.

1936. Restoration of hemolysin-producing
capacity in X-irradiated rabbits. BERNARD
N. JARosLow* AND WiiiiamM H. TALiaFERRO.
Div. of Biological and Med. Research, Argonne

FEDERATION PROCEEDINGS

Volume 16

Natl. Lab., Lemont, and Dept. of Microbiology,

Univ. of Chicago, Chicago, Ill.

The markedly decreased peak hemolysin titer
in rabbits irradiated with 400 r one day before the
intravenous injection of sheep red cells was found
earlier to be partially restored to normal values
when rabbit spleen mince was mixed with the anti-
gen just prior to injection. It was fully restored
when HeLa cell mince, HeLa cell extract or yeast
autolysate, was used instead. The rabbit spleen
mince was ineffective when injected immediately
after irradiation, i.e. one day before antigen in-
jection. (J. Infect. Dis. 1956, in press). The active
principle has now been found to be associated
with the nondialyzable portion of an aqueous
extract of yeast autolysate. The data are con-
sistent with the view that the active principle
does not primarily accelerate recovery of the
immune mechanism, but reacts on the antigen
in such a way that the immune response is initiated
in the x-rayed animal. (Work performed under the
auspices of the Atomic Energy Commission.)

1937. Association of APC virus type 8 with epi-
demic keratoconjunctivitis. EK. Jawertz, L.
Hanna,* P. TuyGeson,* A. NICHOLAS,* AND §.
J. Kimura.* Univ. of California School of Medi-
cine, San Francisco.

Epidemic keratoconjunctivitis (EKC) is an
eye infection with sharply defined clinical charae-
teristics. In spite of conflicting claims, the etiology
of the disease is not established at present. Froma
typical case of EKC we isolated a virus in tissue
culture which now forms the prototype strain of
APC virus type 8. To investigate the possible
relationship of this virus to EKC a survey for
neutralizing antibodies was undertaken. APC
virus type 8 (50-100 TC 50) was mixed with serum
dilutions, left at room temperature for 45 min.
and inoculated inte twice-washed HeLa cell cul-
tures in a volume of 0.2 ml/tube, to which 08
ml of 10% chick serum in mixture 199 was added.
The tubes were incubated in a stationary position
at 36°C and readings for cytopathogenic effects
taken for at least 4 days after the control tubes
had degenerated. The results were considered to
show neutralization when there was a difference
of +++ in readings of experimental and control
tubes for at least 2 consecutive days. Greater than
4-fold rises in neutralizing antibody titers were
seen in 16 of 18 paired sera from patients with
typical EKC in the United States, Canada and
Japan. Heterotypic antibody rises to APC type
3 were 2-fold or less in such patients. Of 70 patients
known to have had typical EKC between 1951
and 1955 in the United States, Canada, Italy or
Japan, 65 (93%) had neutralizing antibodies to
APC 8 virus in a serum dilution of 1:10 or greater.
In 104 control sera from patients with uveitis,
various forms of conjunctivitis and keratitis

‘olume 1§
robiology,

sin titer
efore the
‘as found
al values
the anti-
restored
or yeast
it spleen
1ediately
tigen in-
he active
sociated
aqueous
are con-
principle
r of the
antigen
initiated
inder the
ssion.)

‘ith epi-
vETZ, L,
* anp §.
of Medi-

) is an
| charae-
etiology
. Froma
in tissue
strain of
possible
rvey for
n. APC
th serum
45 min.
cell cul-
hich 08
s added.
position
c effects
ol tubes
dered to
ifference
| control
ter than
ers were
nts with
ada and
PC type
patients
2en 1951
Italy or
odies to
greater.
uveitis,
keratitis

March 1956

(including, herpetic and APC types 2, 3 and 6),
and miscellaneous illnesses not involving the eye
there were 8 (7.7%) that neutralized APC 8 virus
ina dilution of 1:10. It is concluded that infection
with APC virus type 8 is regularly associated with
clinical EKC, although its etiologic role is not
established.

1938. Immunologic significance of antigenic
differences among strains of influenza virus
isolated during same epidemic. Keitu E.
JENSEN. Univ. of Michigan, Ann Arbor.

Strains of type A influenza virus which have
been isolated during several epidemics were com-
pared serologically. Although the majority of
strains isolated in any epidemic year show sero-
logic cross reactions with a prototype strain,
antigenic dissimilarities can be recognized. In
some cases, greater antigenic differences may be
observed among strains isolated during the same
year than that seen with strains isolated at inter-
vals of many years. Antigenic compositions of
several strains obtained in 1951 were determined
by absorptions of ferret sera and compared with
the antibody levels in sera obtained from the virus
donors before and after illness. Results indicate
that serum antibody levels for specific strains
prior to infection did not appear to influence
markedly the antigenic configuration of the strain
isolated. Antigenic characteristics defined for the
virus isolate did not correlate well with antibody
titer increases in sera from the patients. The
variation in antibody response appears attribut-
able to individual differences reflecting varied
histories of antigenic experience with strains of
influenza virus. The immunological significance
of antigenically different strains, as described by
results with animal sera, has been assayed by
testing postvaccination serum pools from 3 age
groups. Results suggest that immunological
reactions of human populations minimize anti-
genic differences and antibody is produced which
reacts with any member of large families of strains
within that type.

1939. Establishment of two lines of nasal ‘epi-
thelial’ cells in continuous culture. WIL-
LIAM S. JORDAN, JR. Dept. of Preventive Medi-
cine, Western Reserve Univ. School of Medicine,
Cleveland, Ohio.

Fragments of human nasal mucosa have been
cultivated in roller tubes for studies of the com-
mon cold. Tissues from 2 donors in homologous
sera produced outgrowths of ‘fibroblasts’ that
were subcultured at 24 and 28 days, respectively.
Subsequent subcultures in medium containing 40%
of pooled heterologous sera were made at 6-12-day
intervals. Following the 3rd subculture, 47-52
days from date of original explant, islands of
‘epithelial’ cells were noted. These cells filled the

AMERICAN ASSOCIATION OF IMMUNOLOGISTS

595

gaps between the fibroblasts in 10-14 days; the
small sheets of cells were removed from the tubes,
tyrpsinized and placed in small tubes. ‘Epithelial’
sheets were formed in 10-14 days, and 2 lines of
cells, DHov and DMB, have been maintained
through 28 subcultures for 9 months. The ‘fibro-
blasts’ eventually failed on subculture, but it
was found that they could be infected by ARD
(RI-67) virus. The ‘epithelial’ cells can be dis-
tinguished morphologically from each other and
from HeLa cells. Both lines of cells permit the
multiplication of poliovirus and ARD viruses,
apparently differ as regards herpes virus and
support the growth of ECHO, Coxsackie and in-
fluenza viruses poorly or not at all. Nasal washings
from patients with colds passed in these cells have
induced no cytopathogenic changes. Both DHov
and DMB cells now have growth rates comparable
to those of HeLa «nd Detroit 6 cells, and stained
preparations exhibit the characteristic features
of malignant cells.

1940. Different intensities of Arthus reaction
in different skin areas of rabbits. REUBEN
L. Kaun, Irvine M. Buatrt,* anp Sun Hyoo
Krim.* Serological Lab. and Dept. of Otolaryn-
gology, Univ. Hosp., Univ. of Michigan, Ann
Arbor.

While investigating the phenomenon of localiza-
tion of protein antigen in different skin-injected
areas of specifically immunized rabbits, it was
observed that, under certain experimental condi-
tions, the intensity of the Arthus reaction to the
injection of the antigen may be marked in one
area and slight in another. The studies were under-
taken primarily to attempt to understand the
immunologic role of the facial skin in the disease
known as midline facial granulomatous ulcera-
tion. The experimental plan was to immunize a
group of rabbits to horse serum and then de-
termine the extent of the local tissue response
(Arthus reaction) to the subcutaneous injection of
the same challenging dose of the antigen in the
midfacial area close to the midline of the face,
and in the abdomen, side and groin of the same
rabbit. The dose used consisted of either 0.1, 0.2
or 0.3 ml of horse serum. In the facial area, the
response to the subcutaneous injection commonly
had the appearance of a severe Arthus reaction,
with marked swelling, induration and with a
central necrotic zone, while in the other injected
areas, the response was generally less severe, often
with no induration and with little or no necrotic
centers. The Arthus reaction is widely believed
to be the result of the union of circulating pre-
cipitins with locally injected antigen. Based on
this view, it might have been expected that the
degree of intensity of the Arthus reaction in all
skin-injected areas would be the same.

596

1941. Effect of antibodies on nucleated mam-
malian cells in vitro. BERNARD KALFaYyAN*
(introduced by JoHN Suae). Beekman-Down-
town Hosp., New York City.

The union between cells and specific antibodies,
in vitro, results, under suitable conditions, in an
observable reaction, such as agglutination, lysis
or nonlytic degeneration of cells. Unlike bacterial
cells, erythrocytes or certain other cells, nucleated
mammalian cells have not been utilized in study
of these reactions as problems in immunology.
During recent studies with different objectives, it
became evident that individually suspended
nucleated mammalian cells could be agglutinated,
lysed or destroyed without lysis, by means of
antibodies of various sorts. Agglutination and
nonlytic degeneration of cells were demonstrated
by incubating freshly procured human leukemia
cells or normal leukocytes with serum of rabbits
immunized by repeated injections of human
tissues, normal and neoplastic. If constant
amounts of cells suspended in isotonic saline,
were incubated at 37°C with increasing dilutions
of inactivated immune serum, the cells were
readily agglutinated, often in mixture with 1:64-
1:128 dilutions of serum. If the cells were sus-
pended in Ringer’s solution buffered at pu
7.0-7.4 and were then incubated with the immune
serum in the presence of complement, the cells
were promptly destroyed as evidenced by irre-
versible degenerative changes. The most striking
change, pyknosis of nucleus, was well developed
within 10-15 min. of incubation; the cytoplasm of
altered cells was moderately swollen and acido-
philic, but was not lysed following prolonged
incubation with the immune factors. Serum of
normal rabbits tested concurrently failed to
agglutinate or destroy the cells. A lytic type of
degenerative change was previously demon-
strated (KALFAYAN AND Kipp. J. Exper. Med.
97: 145, 1953) by incubating individually sus-
pended Brown-Pearce carcinoma cells with the
specific Brown-Pearce antibody in the presence
of complement. Here, the structural changes
were characterized primarily by prompt swelling
and dissolution of cytoplasm, the nucleus remain-
ing relatively normal and the plasma membrane
unlysed following prolonged incubation.

1942. Production of autoimmune bodies to
heart tissue in rabbits inoculated with
streptococcal cultures; nature and histo-
logic localization of the reactive antigen.
Me vin H. Kapuan (introduced by ALBERT H.
Coons). House of the Good Samaritan, Children’s
Med. Ctr., and Dept. of Bacteriology and Im-
munology, Harvard Med. School, Boston, Mass.
The inoculation of group A streptococcal cul-

tures intradermally in rabbits was found to elicit

the appearance in the serum of complement-fixing

FEDERATION PROCEEDINGS

Volume 16

antibodies to normal rabbit heart tissue suspen-
sions. Antibody titers reached a peak within 2 wk,
after the inoculation and rarely persisted longer
than 3-4 wk. Repeated monthly injections for 1 yr,
with small doses of streptococcal cultures of the
same type or of successively different types, by
either the intradermal or intravenous routes,
resulted in each case with a rapid rise and fall of
antibody after each injection, and only rarely ing
sustained elevated level. Heat-killed cultures also
evoked antibody responses, which frequently were
more marked than with live cultures. Culture
filtrates or beef-heart medium alone were usually
ineffective, but in a few animals, small transient
rises were produced. When the experiments were
carried out with streptococci grown in a medium
derived from a digest of casein and soy bean
(trypticase-soy broth), antibodies to heart tissue
were not found. These results have suggested that
the active antigen eliciting the production of
antibodies reactive with rabbit heart is a haptene
with organ specific properties present in the beef
heart medium and absorbed to bacterial cells
during growth. Chemical studies have thus far
identified the substance in rabbit heart tissue and
in beef heart medium as alcohol and ether soluble,
precipitable by barium salts and presumably
associated with phospholipid. Immunohisto-
chemical identification of the antigen in rabbit
heart sections by a modified fluorescent antibody
technique has revealed it within the sarcoplasm of
the myocardial cell contiguous to the cell mem-
brane, and showing a distribution similar to that
to myocardial cell lipid as detected by Sudan
black, or phosphine 3 R stains.

1943. Differentiation of antilipids occurring
in leprosy, lupus erythematosus and syph-
ilis. Joun F. Kent, James C. Burke,* Anp A,
Garcia OTERo.* Depts. of Serology, Walter Reed
Army Inst. of Research, Washington, D. C., and
Univ. Hosp., Havana, Cuba.

Antibodies which react with lipid (cardiolipin-
lecithin-cholesterol) antigens are encountered in
infections and disorders other than the trepone-
matoses. The pattern of their reactions with
antigens of varied lecithin content was studied as
a basis for differentiating them from the antilipids
occurring in syphilis. The present report is based
on sera from patients with leprosy or lupus
erythematosus that reacted with lipid antgens;
all were negative in tests for treponemal im-
mobilizing antibody. The syphilitic sera wer
from authenticated cases of Treponema pallidum
infection. Antigens used in the flocculation tests
contained cardiolipin 0.03%, cholesterol 0.90%
and lecithin in concentrations from 0.15 to 0.30%}
those used for complement fixation contained
cardiolipin 0.0175% and cholesterol 0.30%, com-
bined with 0.022 to 0.175% lecithin. In the floccula-

ots se f&ises at ae a Aste Se Ee eH as OO ehllhlUcceFlUrhrOlUlUrhlUr OOM

wreeo™ +

<za.s8

lume 1§

suspen-
in 2 wk,
| longer
for 1 yr,
s of the
pes, by
routes,
d fall of
rely ing
res also
tly were
Culture
usually
ransient
its were
medium
»y bean
t tissue
ted that
‘tion of
haptene
the beef
ial cells
shus far
ssue and
soluble,
sumably
nohisto-
1 rabbit
ntibody
plasm of
11 mem-
to that
r Sudan

curring
d syph-
* AND A,
lter Reed

C., and

diolipin-
tered in
trepone-
ns with
udied as
ntilipids
is based
yr lupus
antgens;
mal im-
ra were
pallidum
ion tests
1 0.90%
o 0.30%}
ontained
%, com-
floccula-

March 1956

tion reactions, sera from patients with leprosy
or lupus erythematosus reacted to maximal degree
with the antigen of lowest lecithin content (0.15%)
and could be differentiated on this basis from
syphilitic sera which reacted maximally with
antigens containing higher concentrations of
lecithin (0.27-0.30%). The complement fixation
reactions were of no differential value, sera from
persons with leprosy, lupus erythematosus, and
syphilis exhibiting essentially the same reaction
pattern, i.e. all reacted maximally with antigen
of low lecithin content (0.022%). The observations
have direct applications in the standardization
of cardiolipin antigens and simplify differentiation
of the antilipids due to at least 2 nontreponemal
diseases.

1944. Cortisone modification of the dynamics
of influenza virus increase. Epwin D. KIt-
BOURNE. Div. of Virus Research, Dept. of Public
Health and Preventive Medicine, Cornell Univ.
Medical College, New York City.

Systematic investigation of the phenomenon of
cortisone-induced influenza B virus increase led
to repeated observations of an initial lag in the
viral incremental curve in cortisone-injected chick
embryos and tissue culture. This apparent dis-
crepancy from the increased yields of virus finally
attained with cortisone prompted study of the
viral concentrations within the chorioallantoic
membrane—the site of virus formation. Mem-
braines were homogenized in a high speed mixer
and the resulting suspensions treated with RDE
to release virus from cellular debris and inhibitor.
Comparison of membrane virus concentrations
with those of concomitantly harvested allantoic or
tissue culture fluids has demonstrated retention of
virus in cortisone-treated membranes in the early
phase of the incremental curve. Total (membrane
and fluid) virus was found to be increased at all
phases of viral increase. Delayed release of virus
has also been demonstrated in vitro with mem-
brane and RBC suspensions treated with
cortisone. The phenomenon of delayed viral
telease may be explained as an effect of cortisone
1) on cell permeability, 2) directly on cell re-
ceptors or 3) on the concentration of virus-binding
inhibitor within the chorioallantoic membrane.
No effect of cortisone on membrane inhibitor con-
centration has been demonstrable. Present evi-
dence favors an effect on cell permeability in
view of previously recognized reduction of capil-
lary permeability by cortisone and published
evidence by the author that protein exudation
from infected membrane cells is decreased. The
delayed elution from RBC suggests that changes
of the adsorptive state of the cell surface may also
be involved.

1945. A tissue fraction which agglutinates
autologous red cells. Ropert R. KoHn anp

AMERICAN ASSOCIATION OF IMMUNOLOGISTS

597

Epwarp R. ArquiLua (introduced by Louis
PILLEMER). Benjamin Rose Hosp. and Dept. of
Anatomy, Western Reserve Univ. School of Medi-
cine, Cleveland, Ohio.

Homogenates of perfused rat, human and guinea
pig livers agglutinate washed autologous red cells.
The factor or factors responsible for agglutination
were partially purified by dialyzing a buffer ex-
tract of an acetone powder of liver against dis-
tilled water. The active principle was found in the
euglobulin precipitate after dialysis. The partially
purified agglutinating activity was stable at 56°C.
for 2 hr. and boiling for 5 min., whereas cruder
preparations were inactivated by 56°C. for 30
min. Agglutination occurred at 6°C., room tem-
perature and 37°C. Lysis of cells did not occur at
37°C. in the presence of fresh guinea pig serum.
Hemagglutination in vitro could be completely
inhibited by high dilutions of autologous serum.
Agglutinating activity was high in rat liver and
kidney, but not present in significant amounts of
lung, skeletal muscle or spleen (spleen could not
be adequately perfused). High dilutions of rat
liver preparations agglutinated rat, rabbit and
mouse erythrocytes but not red cells from sheep,
human, dog or guinea pig. The ability of human
and guinea pig liver preparations to agglutinate
autologous erythrocytes was much less on a wet
weight basis than that observed in rat liver pre-
pared in the same manner and tested against
autologous cells.

1946. Changed susceptibility to viral infec-
tion in ascites tumor cells of the same
origin. H1nary Koprowsk1, Gait THEIS* AND
Rosert Love.* Viral and Rickettsial Section,
American Cyanamid Co., Research Div., Lederle
Labs., Pearl River, N. Y.

Three ascites tumors—2 mouse lymphomas and

1 rat hepatoma—were adapted to grow progres-

sively in a hitherto insusceptible mouse host.

Tumors of each derivative line were characterized

by greater cellular volume than the original tumor,

by different chromosome ploidy and by higher con-
tent of DNA and RNA per cell. All 3 original
tumors were resistant to infection with viruses
which had been found to multiply in such tumors
as Ehriich or Krebs ascites, MC1M rhabdomyo-
sarcoma ascites, sarcoma 37, etc. In contrast,
tumors of the 3 sublines were susceptible to infec-
tion not only with those viruses previously known
to be oncolytic—Mengo, Bunyamwera and West

Nile—but also with Semliki Forest and western

and eastern equine encephalomyelitis. The latter

3 agents, which oncolyzed cells of the derivative

tumor lines, did not multiply in the neoplastic

cells of any other ascites tumors.

1947. Antigenic relationship of gamma
globulins, cryoglobulins, and macro-cryo-

598

globulin. LEoNHARD KorNGo.p. Sloan-Ketter-

ing Inst. for Cancer Research, New York City.

The elevated serum globulins of patients with
multiple myeloma are antigenically related to
gamma-2 globulin (KoRNGOLD AND Lipari. Cancer
9: 183, 1956). Recently it has been possible to
compare a macro-cryoglobulin with multiple mye-
loma cryoglobulins. The macro-cryoglobulin was
purified by precipitation in the cold. An electro-
phoretically homogeneous gamma-l globulin was
obtained with a major ultracentrifugal component
of 19 Svedberg (S). Antisera prepared by im-
munizing rabbits with human fraction IT (Squibb)
were used for the immunological analysis. Test
antigens were gamma-2 (78) and gamma-l (7S
and 198) globulins, multiple myeloma cryoglobu-
lins, and the macro-cryoglobulin. Immunological
analysis was performed by the Ouchterlony gel
diffusion technique. One precipitin line was formed
by the gamma-2 globulin, and each multiple
myeloma cryoglobulin precipitin line gave a
pattern characteristic of a cross-reaction with it.
Two lines were formed with gamma-l globulin;
one line coalesced with the gamma-2 globulin line
(reaction of identity), the other coalesced with the
single macro-cryoglobulin line. Multiple myeloma
cryoglobulins were antigenically unrelated to the
macro-cryoglobulin. Antibody against gamma-2
globulin was removed by absorption; the absorbed
antisera still reacted with gamma-1 globulin (one
line) and the macro-cryoglobulin, but not with the
multiple myeloma cryoglobulins or gamma-2
globulin. Gamma-l globulin therefore contains 2
antigenically unrelated proteins, one (78) is anti-
genically identical with gamma-2 globulin (7S)
and related to multiple myeloma proteins, the
other (19S) is related to the macro-cryoglobulin.

1948. Changes in properties of human diph-
theria antitoxins associated with use of
chemical agents. WiLuiamM J. Kuuns. Dept. of
Pathology, Univ. of Pittsburgh, Pa.

When diphtheria antitoxin is separated from
other serum components in the course of purifica-
tion, some of its immunological properties may
be altered depending upon the nature of the pro-
cedure. In the present experiments the effects of
some conditions of pH and ionic strength were
investigated. Precipitating and nonprecipitating
skin sensitizing antitoxins utilized in these studies
exhibited immunological criteria which char-
acterize single antigen-antibody systems. When
precipitating antitoxic sera were dialyzed against
distilled water at 4°C and the resulting fractions
tested for antitoxin, activity was found to be
partitioned largely in the euglobulin fraction. In
contrast, most of the neutralizing activity of non-
precipitating antitoxin as detected by rabbit skin
test was contained in the pseudoglobulin. Buffers
of different strengths were employed in other

FEDERATION PROCEEDINGS

Volume 15

dialysis experiments. At 0.01 ionic strength the
relative amounts of precipitating and skin sensi-
tizing antitoxin separated into euglobulin and
pseudoglobulin fractions was similar to that noted
above. The results of passive transfer experiments
indicated that fractions of skin sensitizing anti-
toxic sera dialyzed against either distilled water
or buffers at 0.01 ionic strength no longer retained
wheal and erythema activity. This change was not
observed when the same sera were dialyzed at
0.1 ionic strength and pu 6.0-8.6. The pre-
cipitability of precipitating antitoxin was not
affected by exposure to buffer at px 6.0 and 0.01
ionic strength, but was impaired following dialysis
at pH 4.5 and 0.1 ionic strength.

1949. Increased resistance to infection de-
veloped rapidly after administration of
bacterial lipopolysaccharides. Maurice
Lanpy. Div. of Immunology, Walter Reed Army
Inst. of Research, Washington, D. C.

In addition to the bacterial components re-
sponsible for specific immunity, it is recognized
that certain bacterial products may stimulate non-
specific general defense mechanisms of the host.
However, the full significance of this phenomenon
has not been clearly defined. In the present study,
lipopolysaccharides (endotoxins) isolated from
various gram-negative bacterial species were
found to evoke in mice, an early, nonspecific,
transitory resistance to infection with Salmonella
typhosa. Thus, lipopolysaccharides derived from
smooth and certain rough strains of Escherichia,
Hemophilus, Pseudomonas, Salmonella, Serratia
and Shigella, injected i.p. or i.v., in a dose range
of 1-100 ug, were effective in protecting mice
against challenge with 50 million organisms of
S. typhosa Ty2 in saline (LD59 approximately 10
million). For example, groups of mice, injected
with 10 ug of lipopolysaccharide, at varying time
intervals prior to challenge, reacted as follows
when challenged: 1 hr., fully susceptible; 3 hr.,
significant protection; 6-12 hr., increasing pro-
tection with few deaths; 12-24 hr., complete pro-
tection; 48-120 hr., decreasing protection to full
susceptibility. There appears to be a relationship
between the increased resistance observed in mice
and their elevated properdin titers following in-
jection of bacterial lipopolysaccharides (LANDY
AND PILLEMER, to be published). The early in-
crease in resistance, following injection of lipo-
polysaccharide, was also observed in mice
experimentally infected with E. coli, Proteus
vulgaris, and Ps. aeruginosa; however, the time,
dose and duration relationships for resistance to
these infection systems were significantly different
from the pattern observed with S. typhosa.

1950. Immunochemical studies with hemo-
eyanin. Cuartes A. Leone. Argonne Nall.
Lab., Lemont, Ill.

ET NE ee Gk as ee ey ie ae

plume 15

gth the
nD sensi-
lin and
at noted
riments
ng anti-
d water
retained
was not
yzed at
he pre-
was not
and 0.01
‘dialysis

ion de-
tion of
VA URICE
ed Army

ents re-
cognized
late non-
the host.
10menon
it study,
ed from
es were
ispecific,
ilmonella
red from
herichia,
Serratia
se range
ng mice
nisms of
ately 10
injected
ring time
; follows
le; 3 hr,
sing pro-
ete pro-
n to full
ationship
d in mice
wing in-
| (LANDY
early in-
. of lipo-
in mice

Proteus
the time,
stance to
- different
sa.

1 hemo-
une Nall.

March 1956

Purified solutions of hemocyanin from the crab,
Cancer magister, were fractionated with Na.SO,
to yield 4 populations of molecules. Electro-
phoretic and ultracentrifugal analyses of the
preparations indicated the presence of 3 distinct
kinds of molecules among the 4 fractions. Quanti-
tative precipitin tests, both gravimetric and
turbidimetric, failed to reveal any significant
serological differences among the 4 preparations.
Agar-plate precipitin tests showed that each of
the 4 fractions, while sharing molecules in com-
mon, had serologically unique molecules. A total
of 7 distinct zones of reaction were detected in the
agar-plate tests. The agar-plate precipitin test is
more sensitive than other precipitin tests for de-
tecting serologically discrete molecules. The
physicochemical properties of the different protein
molecules in the original solution of hemocyanin
were too similar to permit clean separation using
the Na2SO, fractionation. (Work performed under
the auspices of the Atomic Energy Commission.)

1951. Effect of focal irradiation on antibody
production. SipNEY LeskowITz AND JOHN B.
GRAHAM (introduced by Byron WaAKSMAN).
Massachusetts General Hosp., Boston.

In an attempt to develop a more rational ap-
proach towards radiotherapy in cancer, a study
was made of the effect of local x-irradiation of an
antigen depot on antibody production. Sheep
erythrocytes were injected intracutaneously into
rabbits and the site of injection given 1000 r by a
Van de Graaff generator. Bleedings taken 2 wk.
later showed slightly higher titers of hemolysin
in the irradiated animals than in the controls.
To obtain more quantitative results, the work
was repeated using alum precipitated diphtheria
toxoid as antigen and titrating the antitoxin pro-
duced by toxin neutralization tests in rabbit skin.
Rabbits were injected intracutaneously with the
toxoid and some were irradiated at the site of
injection. Twenty days later, all animals were bled
and patches of skin around the site of injection
were excised and extracted with saline. Titration
of the sera showed modest increases in circulating
antitoxin over the controls and titrations of the
skin extracts showed considerably larger amounts
of antitoxin in the irradiated patches of skin.
Focal irradiation seems to result in an enhance-
ment of antibody production in contrast to its
suppression by whole body irradiation. (Sup-
ported in part by American Cancer Society.)

1952. Immunochemical studies of the kinetics
of monomerization of human serum al-
bumin mercury-dimer. LAWRENCE LEVINE,
RosBertT SMITH AND Ray K. Brown (introduced
by GitBertT Datuporr). Div. of Labs. and Re-
search, New York State Dept. of Health, Albany.
The reaction of crystallized serum-albumin

mercury-dimer (HSA—S—Hg—S—HSA), its mer-

AMERICAN ASSOCIATION OF IMMUNOLOGISTS

599

cury monomer (HSASHgX), and decanol crystal-
lized HSA with antisera to HSASHgX was
analyzed by the quantitative precipitin technique.
No antigenic differences were found between
decanol crystallized HSA and HSASHgX as
judged by this method. In the reaction of dimer
and antimonomer, the equivalence zone was
shifted towards the zone of antigen excess and
antigen excess inhibition was less marked.
Analyses of the precipitates in antibody excess
indicate that 2 antigenic sites were occluded as a
result of dimerization. The difference in nitrogen
precipitated by monomer and dimer in antigen
excess was used to study the rate of the reaction
HSA—S—Hg—S—HSA + Hgt+ — 2HSASHgxX.
Under the conditions used, the antigen-antibody
reaction was more rapid than the monomerization
reaction and effectively stopped further interac-
tion of Hgt* with the dimer. Hg**, in the concen-
trations used, had little effect on dimer once the
antigen had reacted with the antibody. The aver-
age reaction rate constant at 0°C over a 10-fold
change in initial mercury concentration was
1.83 X 105 1/m sec. The rates at 9.8°C and 20.1°C
were 4.45 X 105 1/m sec. and 10.2 X 1051/m sec.,
respectively. Rate constants are in terms of con-
centration of mercuric ion added and should be
nearly proportional to those calculated on a
mercuric ion activity basis. The energy of activa-
tion was 11,600 cal/m.

1953. Effect of antibiotics on antigenic struc-
ture of Shigella flexneri. Rosert B. LINDBERG
AND Korsone Li (introduced by Maurice
Lanpy). Walter Reed Army Inst. of Research,
Washington, D. C., and the 406th Med. General
Lab., Tokyo, Japan.

Shigella flexneri strains are found in the specific
phase at the time of isolation from cases of dysen-
tery. The nonspecific phase ordinarily is encoun-
tered when, during maintenance in culture,
specific antigenic components disappear. This
may happen at varying times following isolation.
However, factors influencing this change are
rather obscure and many strains can be main-
tained indefinitely in the specific phase. When
such specific phase strains of S. flexneri types la,
2a and 4a were exposed to subinhibitory concen-
trations of oxytetracycline, chlortetracycline,
chloramphenicol and streptomycin, nonspecific
phase variants usually appeared promptly. How-
ever, with certain strains of S. flexneri 2a and 4a,
this change did not occur following exposure to
chlortetracycline, although the other antibiotics
did effect a change in phase. Paradoxically, after
such treatment with chlortetracycline, cultures
were fixed in the specific phase and subsequent
exposure to the other antibiotics failed to incite

reversion. Alteration in the antigenic structure of

dysentery bacilli by antibiotic at levels com-

600

parable to those attained in clinical treatment may
have a bearing on the epidemiology of shigellosis.

1954. A study of Canine distemper in ferrets
and dogs by means of fluorescent antibody.
Cui’en Liv anp Davin L. Corrin.* Depts. of
Bacteriology and Immunology and of Pathology,
Harvard Med. School, and Dept. of Pathology,
Angell Memorial Hosp., Boston, Mass. i
Using the fluorescent antibody technique

(Coons AND Kaptan. J. Exper. Med. 91: 1, 1950)

a study of the pathogenesis of canine distemper

infection in ferrets showed that viral antigens

were first detectable in cervical lymph nodes 2

days after intranasal inoculation. Subsequently,

the virus spread to the mediastinal cells, the

mesenteric nodes and the spleen. After about 1

wk. of viral multiplication in the lymphoid and

the reticuloendothelial cells, viral antigens began
to appear in the epithelium of the gastrointestinal
tract, the respiratory tract, the urinary tract
and the cutaneous tissues. Viremia was present
before fever and other clinical signs of distemper
infection appeared, and persisted until the death
of the animals. A rapid diagnosis of distemper
infection in ferrets could be made from the blood
smears. The distribution of distemper viral anti-
gens in naturally infected dogs was similar to that
seen in experimentally infected ferrets. However,
viral antigens were also found in the neurons, the
astrocytes and in the blood vessel walls in brain
sections of dogs with central nervous system dis-
temper. In dogs, acute distemper infection could
be diagnosed from conjunctival smears. The viral
antigens usually appeared as fine intracytoplasmic
fluoroescent granules during early infection. As
infection progressed, the antigens aggregated to
form large oval objects in the cytoplasm. In identi-
eal cells, the viral antigens demonstrated by
fluorescent antibody staining were found to corre-
spond to the inclusion bodies stained by the Seller
method which confirms the finding reported by
Moulton and Brown (Proc. Soc. Exper. Biol. &
Med., 86: 99, 1954).

1955. Polt®myelitis virus complement-fixing
antigen extracted from human feces with
the use of ion-exchange resins. GERALD A.
LoGripro. Dept. of Labs., Henry Ford Hosp.,
Detroit, Mich.

The extraction of poliomyelitis virus from
human feces employs the same principles as those
reported in the procedure for partial purification
and concentration of poliomyelitis virusfrom mouse
infected brain tissue. It was necessary to change
the technique from an open system to a closed one
for reasons of safety and to alter the combination
of resins to one more suitable to feces. In step J
of the procedure, 20 gm of feces in 4 1. of distilled
water is passed through a monobed resin column
containing 350 gm amberlite IRA-410 (strong base

FEDERATION PROCEEDINGS

Volume 16

anion exchange resin, bead size) and 350 gm of
amberlite IRC-50 (weak acid cation exchange
resin, bead size). In this step partial demineraliza-
tion is accomplished, the effluent remaining above
pH 5.0 with the virus retained in the effluent.
In step II, 100 gm of amberlite XE-67 (strong base
anion exchange resin, very fine powdered form)
is added to the effluent from step J and allowed to
react for 30 min. in a batch procedure. The resin-
virus-protein complex is recovered in a specially
designed glass cylinder and the effluent discarded,
In step III, the resin-virus-protein complex is
then treated in the same cylinder with 400 ml of
10% disodium acid phosphate for 30 min. While
the resin is still retained in the cylinder, the
phosphate-virus eluate is drained through the
stopcock of the cylinder. The phosphate-virus
eluate is dialyzed against distilled water to free
it of phosphate and then centrifuged at 40,000 rpm
in the ultracentrifuge. The sediment is re-
suspended in 20 ml of veronal buffer and used in a
complement fixation test with known monkey
antipoliomyelitis sera. This procedure is being
compared with the tissue culture technique of
virus isolation.

1956. Single-visit immunization with emulsi-
fied pollen antigen (Freund’s adjuvant).
Mary H. Lovetess. New York Hosp. and Dept.
of Medicine, Cornell Univ. Med. Colleges New
York City.

Freund’s water-in-petrolatum emulsion has been
adapted to the preseasonal immunization of over
100 ragweed pollen-allergic subjects during the
past 8 yr. Release from a subcutaneous depot has
been found so delayed that patients tolerate per-
haps 100 times more antigen than in unemulsified
form. This has enabled us to give, during a single
visit, sufficient allergen to duplicate the clinical
and immunologic (ophthalmic) results of conven-
tional, multivisit courses. The emulsion contains
mineral oil, falba and aqueous pollen extract,
but no acid-fast bacilli. A volume of 0.5, 0.75 or
1.0 ml is given, depending on whether preliminary
instillation tests of the conjunctive indicate that
the patient has marked, average or below average
allergy. The total volume is given in several frac-
tional injections into the same area, at 4] hr. inter-
vals. It is administered 2-3 months prior to
pollination. The risk of untoward reaction to
emulsified allergen is no greater statistically than
to unemulsified antigen in standard: (much
smaller) dosage. When vegetable oil was substi-
tuted for mineral oil, allergen was more rapidly
released as indicated by an increased incidence
of overdose reactions and an earlier appearance
of manifestations. Clinical and immunologic
(conjunctival) results were not significantly al-
tered from those associated with mineral oil
emulsions.

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March 1956

1957. ECHO viruses isolated in survey of nor-
mal persons, Luzon, Philippine Islands.
E. H. Lupwie* anp W. McD. Hammon. Dept.
of Epidemiology and Microbiology, Grad. School
of Public Health, Univ. of Pittsburgh, Pitts-
burgh, Pa.

During attempts to isolate poliovirus from some
2000 rectal swabs collected from 1200 normal
American military personnel and their de-
pendents, and groups of Filipinos and Negritos on
Luzon, 204 agents have been recovered in tissue
cultures prepared from trypsinized monkey
kidney. Of these, 41 are poliovirus. Among the 163
agents not typable as poliovirus at least 4 specific
and different viruses have been identified using
hyperimmune rabbit and monkey sera. The com-
monest type is our prototype 2-165, which is
antigenically related to Melnick’s Farouk, ECHO
type 1. A few are Coxsackie type A-9. Two other
groups have been found dissimilar to agents de-
scribed by others and are identified as Travis
2-85, ECHO type 12, and Hamphill 2-188, ECHO
type 13. Sera of persons from whom some of these
viruses have been isolated, or sera from members
of their families, show good complement fixing
antibody titer rises with antigen prepared from
several of the agents. Neutralizing antibody
against various of these agents has been demon-
strated in American and Japanese gamma
globulin. Neutralizing antibody has also been
shown, sometimes with rising titer, in serial sera
from cases from Luzon diagnosed in 1954 and 1955
as nonparalytic poliomyelitis or aseptic menin-
gitis, but not confirmed as poliomyelitis by labora-
tory methods. Work is continuing on the
identification and classification of these human
enteric viruses.

1958. Effect of triiedothyronine and propyl
thiouracil on native resistance to tuber-
culosis. Max B. Lurie AND GroraE S. Nrnos.*
Henry Phipps Inst., Univ. of Pennsylvania,
Philadelphia.

The BMR of 30 litter mates of the susceptible
CaC race was determined. Ten rabbits served as
untreated controls; 10 were fed propyl thiouracil
which reduced their BMR 25%; 10 were treated
with pu-thyroxine and u-triiodothyronine, suc-
cessively, intramuscularly; this increased their
BMR 40%. The 3 groups were then exposed to
the quantitative inhalation of virulent human
type tubercle bacilli. Thiouracil and _triiodo-
thyronine treatment, respectively, was continued
throughout the course of the infection. The tuber-
culin sensitivity of the hypothyroid rabbits was
markedly reduced, apparently due to the anti-
inflammatory effect of the antithyroid drug, since
the inflammation due to India ink was also sup-
pressed in these rabbits, as in animals under the
influence of cortisone. Five weeks after infection

AMERICAN ASSOCIATION OF IMMUNOLOGISTS

601

the control rabbits showed extensive tuberculous
lesions with numerous bacilli in the far advanced
caseous centers, mature epithelioid cells with few
bacilli, and widespread perifocal granulation
tissue. The tuberculosis in the triiodothyronine
rabbits was either completely suppressed or re-
mained as few barely visible minute nodules with-
out caseation, composed of small collections of
mature epithelioid cells with few or no bacilli,
flanked by round cells. The tuberculin sensitivity
of these rabbits was less than that of the controls.
The lesions in the thiouracil rabbits resembled
those in cortisone treated animals and consisted
of intra-alveolar plugs of incompletely caseous
tissue, teeming with bacilli, surrounded by im-
mature epithelioid cells swarming with micro-
organisms. It is surmised that hyperthyroidism
may increase the physiological activity of the
phagocytes whereas cortisone depresses it by sup-
pressing thyroid function.

1959. Specific enhancement of phagocytic
function independent of humoral media-
tion.* STANLEY Marcus AND Davin M. Donatp-
son. Univ. of Utah College of Medicine, Salt Lake
City, and Brigham Young Univ., Provo, Utah.

A modification of the histologic method devised
by Metchnikoff in studies of phagocyte function
has been developed to measure intracellular diges-
tive (cytopeptic) action of phagocytes. The fol-
lowing results indicate that immunization induced
increase in cytopeptic action is due to cellular
adaptive alterations and not to humoral changes.
The immunization induced increase in cytopeptic
action is reversed by irradiation. However, with
the procedures employed, the specific antibody
concentrations were not significantly different
in groups of irradiated-immunized and immunized
animals. No difference in cytopeptic action was
observed when the phagocyte suspensions from
individual rabbits were divided into 2 aliquots
and placed in the serum of either an irradiated-
immunized rabbit or a nonirradiated-immunized
rabbit. Studies in vitro showed that the source of
phagocytes was of primary importance. Washed
peritoneal phagocytes from normal rabbits (non-
irradiated and nonimmunized) tested for cyto-
peptic capacity in pooled antibody, digested the
cellular antigen significantly less efficiently than
did similarly treated phagocytes from immunized
animals but significantly more efficiently than
phagocytes from either immunized or nonim-
munized irradiated rabbits. Differences in cyto-
peptic action by phagocytes from immunized or
nonimmunized irradiated animals were not sig-
nificant.

1960. Disulfide bonds in bovine y-globulin.
Gasor Marxkus* AND Frep Karusu. Dept. of
Pediatrics, School of Medicine, Univ. of Pennsyl-

602

vania and Children’s Hosp. of Philadelphia,

Philadelphia.

Disulfide bonds were reduced by treatment with
8-mercaptoethylamine-HCl (MEA), 0.001 m-0.2 m,
at pH 7.0 and 25°C. The excess MEA was removed
by passage through columns of Dowex-50. Protein
SH groups were determined by the amperometric
titration method of Benesch and Benesch. Native,
unreduced bovine y-globulin has one SH group
per mole. Reduction of native protein yields 13
SH groups (6 disulfide bonds split). The resulting
protein shows no change in viscosity or optical
rotation, but is more susceptible to acid denatura-
tion than the untreated protein. Bovine y-globulin
denatured in 0.1 m sodium decyl sulfate is com-
pletely reduced in 0.1 m MEA with the appearance
of 37 SH groups (18 disulfide bonds reduced. Re-
duction to the extent of 27 SH groups results in
no significant increase in viscosity. After removal
of the reducing agent there is no change in optical
rotation from the value reached by protein ex-
posed to detergent only. It has not been possible
to establish an equilibrium constant for the re-
duction reaction, either for the native protein or
for the detergent-treated protein. We may con-
clude that 3 of the disulfide bonds in bovine
y-globulin are easily accessible for reduction,
whereas 3 become reactive only after detergent
denaturation.

1961. Cytoplasmic inclusions of HeLa cells in
tissue culture. Beryt T. Mason,* HERMAN C.
Mason AND Murray FRANKLIN.* Dept. of
Neurology, Univ. of Illinois College of Medicine,
Illinois State Psychopathic Inst., and Cook
County Hosp., Chicago.

During studies of Japanese B virus and polio-
myelitis viruses in HeLa cell tissue cultures our
interest was aroused by the occurrence in nutrient
and maintenance solutions of well characterized
basophilic and eosiniphilic cytoplasmic inclusions.
Although easily seen by Giemsa and other stains
they seem to have attracted little attention and
to have escaped recognition of experienced tissue
culture workers. They have not been described
or reported elsewhere and were first detected by
us in HeLa cell studies of Japanese B virus inhibi-
tion of poliomyelitis (Saukett) virus. Normal
HeLa cells in tissue culture do not present intra-
nuclear inclusions. Normal and JBE infected cell
in nutrient and maintenance solutions show the
presence of round basophilic cytoplasmic inclusion
bodies. Normal cells and cells infected with
Japanese B were graded on presence of vacuoles
and the basophilic and eosinophilic staining quali-
ties (Giemsa) of their cytoplasmic inclusions.
Four type inclusions were found in both normal
and JBE infected cells; 7) round basophilic in-
clusions, 2) round eosinophilic inclusions, 3)
basophilic granules with pale blue matrix, 4)

FEDERATION PROCEEDINGS

Volume 16

basophilic granules on an eosinophilic matrix,
These do not appear to be mitotic residues. HeLa
cells infected with JBE and not demonstrating
cytopathogenicity do not consistently show a
greater number of basophilic or evsinophilic in-
clusions. Titration by dermal, intraperitoneal,
intracerebral, corneal, intratesticular and foot
pad routes of infected HeLa cells containing these
inclusions into rabbits, mice and guinea pigs has
not demonstrated the presence of a virus or other
infectious agent.

1962. Growth of Japanese B virus (JBE) in
HeLa cell cultures and ink ‘bition of polio-
myelitis (Saukett) virus. HERMAN C. Mason.
Illinois State Psychopathic Inst., Chicago.

Two lines of JBE (Taira) virus (Army Med.
Service Grad. School) were grown in HeLa cell
tissue cultures; line 1 now in the 41st tissue cul-
ture passage, and line 2 now in 32nd tissue culture
passage, gave mouse LDso titers over 10-45 and
10-3/.03 ml for the 40th and 31st tissue culture
passages, respectively. The peak for JBE virus is
irregularly reached between 48 and 120 hr. A peak
of 10-®-5/.03 ml was reached once at 5 days; the
average titer is 10-5 or 10-*-5/0.03 ml with virus
yields approximately the same in nutrient or main-
tenance solutions. Cytopathogenicity was not
observed except in the 13th and 41st tissue culture
passages of line 1, and not until the 32nd tissue
culture passage for line 2. The cells do not show
the typical degeneration as with poliomyelitis,
Eastern and Western equine, and West Nile
viruses, but after rounding of individual cells,
cells increase in size and perhaps cluster, and over
a period of days slowly separate from the glass,
Not all cells are affected, normal forms remain.
Line 1 gave the greatest effect ; line 2 demonstrated
identical effect but at much slower rate with
fewer cells involved. There is cytopathogenic
inhibition and titer inhibition of poliomyelitis
(Saukett) virus put in contact with JBE infected
cells; cytopathogenic inhibition can be complete,
partial or not present. Poliomyelitis virus is able
to multiply in the presence of JBE virus without
observable destruction of HeLa cells. The inhibi-
tion appears correlated with the early (12 hr.)
titer of JBE virus after the poliomyelitis virus
has been added.

1963. Antigenicity of polyvinylpyrrolidone.
Paut H. Maurer. Dept. of Pathology, Univ. of
Pittsburgh School of Medicine, Pittsburgh, Pa.
The antigenicity of polyvinylpyrrolidone (PVP)

of various molecular sizes has been tested in both

man and rabbits. Microprecipitin techniques as
originally developed by Heidelberger and Mac-

Pherson have been adapted to these studies.

Although various techniques of immunization

with adjuvants were tested no antibody was

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March 1956

detected.in the sera of rabbits. Immunizations in
man were performed by intramuscular injections
of solutions of the PVP. Only PVP of K-87 value
(average molecular weight > 1,000,000) produced
significant amounts of precipitating antibody.
In the individuals studied no skin reactions were
observed. However one volunteer exhibited a
marked systemic reaction after one injection of
PVP. All of the sera studied for passive
anaphylaxis in guinea pigs were negative. Sera
from the best reactors were calibrated by quanti-
tative immunochemical procedures with the
various PVP preparations. The thermal stability
of the antibody in both unfractionated and frac-
tionated sera has been studied. Specific precipi-
tates were soluble not only in excess PVP of any
molecular size, but also in the monomer N-vinyl
pyrrolidone. Additional bleedings were taken
periodically to determine the half-life of the
antibody to PVP. In some volunteers the antibody
persisted for several months, whereas in others
it disappeared with a half-life of about 3 wk.
Some of the applications and implications of these
findings concerning the antigenicity of PVP will
be presented. (Grant from the Office of the Surgeon
General.)

1964. Complement fixation with purified
polio virus. MANFRED M. MAYER AND HERBERT
J. Rapp.* Johns Hopkins Univ. School of Hy-
giene and Public Health, Baltimore, Md.
Complement fixation (C’F) tests with crude

tissue culture virus as antigen have yielded con-

fusing reactions. Thus, it has been noted that in
successive bleedings from polio patients, antibody
titer may rise, or fall, or remain stationary. Some
sera are positive by C’F, though lacking neutraliz-
ing antibody of the corresponding type. In
contrast, with highly purified virus as antigen

(obtained from Drs. Schwerdt and Schaffer of

Berkeley, Calif., and from Dr. Charney of Sharp

and Dohme), antibody titer rises to a peak in 1 or

2 months after onset, followed by slow decline.

Furthermore, all sera showing antibody by C’F

have been found positive by neutralization.

However, there is a large differential in sensitivity

between C’F and neutralization. Thus, sera with

high neutralizing titer are usually positive by

C’F, while sera with low neutralizing titer are

usually negative. These observations suggest:

1) Identity of neutralizing antibody with the anti-

body detected by C’F with purified virus as

antigen. 2) Infected tissue culture fluids, or crude
virus concentrates, contain at least 2 antigens,
viz., intact virus particles and particles of a differ-
ent specificity (possibly virus fragments), which
may be separated by chemical and physical
methods, such as electrophoresis or ultracentrifu-
gation in a sucrose density gradient. 3) Some polio
patients produce antibody to both antigens,

errs i

AMERICAN ASSOCIATION OF IMMUNOLOGISTS

603

which leads to confusing super-imposition of 2
antigen-antibody systems when crude virus
serves as C’F antigen. (Supported by Natl. Fndn.
for Infantile Paralysis.)

1965. Rate of immune response in mice to
enteric vaccines in relation to properdin
levels. Grace L. McCatitum,* Howarp J.
BouHNER* AND GEOFFREY EpsALL. Div. of Im-
munology, Walter Reed Army Inst. of Research,
Washington, D.C.

Typhoid vaccine elicited comparable immune
responses in mice challenged with the homologous
organism 2 or 6 days following vaccination. Mice
inoculated with Shigella flexneri 3 or cholera vac-
cine were not protected against homologous
challenge 2 days post-immunization. An increase
in properdin titer was found in sera of mice inocu-
lated with typhoid vaccine, but not in those in-
jected with flexner or cholera vaccines. Further
investigation of the immune response in mice and
its possible relation to properdin was undertaken
using time intervals between vaccination and
challenge ranging from 1 hr. to 14 days. Consider-
able variation was found in the rate of immunity
conferred by the 3 vaccines: no significant pro-
tection was observed in mice inoculated with
typhoid vaccine through 24 hr. Protection was
marked 48 hr. post-immunization and similar to
that found at further intervals. The pattern with
flexner vaccine was similar except that marked
immune response did not appear until 4 days post-
immunization. A definite response was detected
with cholera vaccine 6 and 24 hr. post-immuniza-
tion. This protection was considerably less at 48
hr., then became marked with a 4-day interval
and continued through 14 days. Zymosan inocu-
lated into mice 2 or 6 days preceding challenge
protected mice against viable S. typhosa. How-
ever, marked protection was found at both inter-
vals in mice inoculated with zymosan preceeding
challenge with Sh. flexneri 3 or V. comma.

1966. Behavior in Coombs test of anti-A and
anti-B produced by immunization with
various A and B substances and by hetero-
specific pregnancy. FRepERIC McDurrin*
AND Etvin A. Kabat. Depts. of Neurology and
Microbiology College of Physicians and Surgeons,
Columbia Univ. Neurological Inst., New York City.
Several workers have reported that more than

one variety of anti-A and anti-B can be distin-

guished by using techniques originally developed
in studying Rh antibodies. However, since all
anti-A and anti-B sera produce agglutination in
saline, these differences have been established in
various ways, one of which is that some sera give

a Coombs titer beyond the saline endpoint while

others do not. The antibodies in the former sera

have been called ‘immune’ by several investigators
and those in the latter ‘natural.’ To test this

604

hypothesis sera were examined from individuals
immunized with A or B antigens or with both,
either by heterospecific pregnancy or by injecting
purified blood group substances. Coombs tests
were performed in block titration using antiserum
to purified y-globulin, and the enhancement of
the saline agglutinin titer, if any, was measured.
The sera studied fell into 2 groups, one in which
enhancement of the saline titer by Coombs serum
occurred and the other in which it did not. Both
types of behavior were shown by antibodies known
to have resulted from immunization. The findings
indicate that the distinction between so-called
‘natural’ and ‘immune’ antibodies is untenable
since antibodies known to be of immune origin
may show either type of behavior. (Supported by
grants from United Cerebral Palsy and the William
J. Matheson Commission.)

1967. Type I poliomyelitis strains in monkeys.
I. W. McLean, Jr., E. A. Trum,* W. A. Ricut-
sEL,* E. Z. Rops* anp H. P. Drosecx.* Re-
search Labs., Parke, Davis and Co., Detroit, Mich.
The following type I strains were studied for

monkey virulence: Pitt. no. 2 Yuhasy; Pitt. no.

10 McGrady; Pitt. no. 15 Coleman; Pitt. no. 18

Bostivick; Pitt. no. 20 Jennings; Charleston 13;

Charleston 16; Charleston 65; W-S 40-Y; Brun-

hilde-Enders-Chimp; Parker; P2149; P2226; P1553,

and Mahoney. A total of 128 rhesus and 189

cynomolgus monkeys were used. All monkeys were

inoculated intramuscularly, right gastrocnemius
muscle, with 2.0 ml. of the strain being studied.

Rhesus monkeys were inoculated with undiluted

suspensions, 9 being inoculated with each of the

strains, except Parker, for which 13 were used;

Pitt. 15, Charleston 16, Brunhilde-Enders, and

W-S were inoculated in 4 rhesus. Mahoney was

not run in rhesus. Ten cynomolgus monkeys

(minimum) were inoculated with each strain, and

5 cynomolgus were inoculated with a 10— dilution

of each strain except Pitt. 15, Charleston 16,

P1553, P2149, Brunhilde-Enders, W-S, and Ma-

honey. A 10~ dilution Parker, Charleston 65,

P2226, and Mahoney strains and 10-* and 10‘

dilutions of the Mahoney strain were inoculated

into 5 more cynomolgus monkeys. All monkeys
on test were checked daily for signs of disease and
were killed at 21 days or earlier if they became
moribund. Blood samples were taken on the 4th
and 6th day following inoculation, and the animals
were bled out before being killed. The 4- and 6-day
samples were titrated in tissue culture for viremia
study, and the final bloods were titrated for
antibody. Central nervous system specimens were
taken from all monkeys for histopathological ex-
amination. Results show that all of the strains
were highly pathogenic for monkeys varying only
slightly in degrees of virulence. Likewise, differ-
ences in antigenic potency were slight. Clinical

FEDERATION PROCEEDINGS

Volume 16

findings were supported closely by the histo-
pathological findings.

1968. Certain factors influencing hemolytic
action of complement. Davin L. McVickar
(introduced by A. F. Rasmussen, Jr.). Dept. of
Infectious Diseases, School of Medicine, Univ. of
California, Los Angeles.

Studies on the uptake of complement (C’) at
low temperatures (0°-4°C) have shown that there
can be marked lysis if erythrocytes sensitized at
amboceptor concentrations greatly increased over
the usual optimal level are used. Increase in ambo-
ceptor concentration is accompanied by an in-
crease in the amount of hemolysis. Since the ad-
sorption of C’1,4 and C’2 is known to take place
at 0°C, the observed results must relate to the
utilization of C’3, which heretofore has been con-
sidered not to be fixed to any appreciable extent
at temperatures lower than 15°C. Data will be pre-
sented from studies carried out with C’3 on the
residual (unlysed) erythrocytes (EAC’1,4,2);
these data indicate that lysis at low temperatures
cannot be explained in terms of increased uptake
of C’1,4,2 at increased amboceptor concentration.

1969. Poliomyelitis: a sero-epidemiologic sur-
vey of remote areas in the Amazon Basin
and Middle East. H. M. Meyer, Jr., N. G.
Rogers AnD D. C. GaspusExK (introduced by
M. R. Hinieman). Walter Reed Army Med.
Center, Washington, and Univ. of Maryland
School of Medicine, Baltimore.

The prevalence of neutralizing antibodies
against poliomyelitis viruses was determined in
sera from 285 infants, children and young adults
living in diverse topographic and _ sociologic
regions in Bolivia, Peru, Afghanistan, Iran and
Turkey. Ninety-three % of Peruvian and Bolivian
4 to 5-year-old villagers had experience with one
or more types of virus, i.e., antibody titers of 1:4
or greater in tissue culture metabolic inhibition
tests. Antibodies for all 3 sero-types were present
in 95% of persons 6 to 20-years-old. Almost identi-
cal results were obtained among urban and vil-
lage Afghans and Iranians. Sixty-eight % of
Turkish infants (8 mo.-2 yr.) showed serologic
evidence of prior poliomyelitis infection while
100% of Turkish 2 to 4-year-olds had antibodies
for at least one type and 57% had all 3 types of
antibodies. These data clearly indicated the high
prevalence of poliomyelitis infection in early life
in these populations in contrast to the delayed
experience of persons living under better sanitary
conditions. By comparison, the occurrence of
complement-fixing antibodies against herpes
simplex and mumps viruses in the groups sur-
veyed did not differ significantly from that found
in this country.

or a ee ee eis ee ls ee Sis “a - ee - a, a Se we

_—

March 1956

1970. Salt-free chromatography of proteins
on cellulose ion exchanges using carbon
dioxide solutions. Miuton A. Mitz anp Sam
S. YANARI (introduced by K. C. Rosstns).
Research Div., Armour and Co., Chicago, Ill.
Chromatography of proteins on cellulose ion

exchangers has been accomplished with a distilled
water-CO: salt-free system. In the fractionation
of kidney cathepsins it was found that the CO,
content of a solution was important in the adsorp-
tion of the proteins on cellulose anion exchangers.
Since weak anion exchangers will not normally re-
move CO, from solution the results were un-
expected. Studies were made at 0 and 25°. The
ease of dissociation of a protein from the exchanger
by COzis different for various proteins. The partial
pressure of carbon dioxide over the solution is an
important factor. Reduction of the partial pres-
sure of CO: over the protein eluate will permit the
readsorption of the protein. Conversely the pro-
tein is not adsorbed if the eluate is freshly treated
with CO. These experiments suggest carbamate
and carbonate formation. A cellulose anion ex-
changer column from which most of the protein
has been desorbed with CO: solution can be reused
after washing with distilled water. The kidney
cathepsin eluate, freed of CO:, had a px which
corresponded to the px obtained after exhausitve
dialysis or by fractionation using electrical trans-
port methods. Studies have been made on hemo-
globin, serum albumin, insulin, pepsin, egg al-
bumin, liver catalase, and nucleic acids.

1971. Biotin in purine biosynthesis. A. G.
Moat, C. N. Wiixins, Jr. AND H. FRIEDMAN
(introduced by A. Bonpt1). Div. of Microbiology,
Hahnemann Med. College, Philadelphia, Pa.
Work currently in progress on the biosynthesis

of purines by yeast has revealed that under the

conditions of biotin deficiency Saccharomyces
cerevisiae accumulates an aromatic amine. This
aromatic amine has been tentatively identified as
4(5)-aminoimiadzole on the basis of its extreme
lability, its ultraviolet absorption spectrum, and
the absorption spectrum exhibited when the com-
pound is diazotized and coupled with an aromatic
color reagent (Bratton-Marshall reaction). This
arylamine does not accumulate in the presence of
concentrations of biotin which are optimal for
growth of the organism. The amount of amino-
imidazole observed can be markedly increased by
the inclusion of methionine or glutamic acid in the
culture medium. Either of these compounds stimu-
lates aminoimidazole production, but the maxi-
mum yield is obtained in the presence of both of
these amino acids. The aromatic amine is produced
by both growing cultures of yeast and by washed
suspensions of biotin-deficient cells. The amount
of aminoimidazole formed is decreased in the
presence of aspartic acid. This correlates with the

AMERICAN ASSOCIATION OF IMMUNOLOGISTS

605

growth-stimulating effect of aspartic acid on this
yeast under the conditions of biotin deficiency.
Purines also prevent the accumulation of the
intermediate. Various factors which affect amino-
imidazole production by S. cerevisiae will be dis-
cussed. (Supported by Grant. No. MET-48 from
the American Cancer Society.)

1972. Structure and development of ARD
(APC) virus in HeLa cells examined in the
electron microscope. CouNcILMAN Moraan,*
CaLDERON Howse, Harry M. Ross anp Dan H.
Moors.* Depts. of Microbiology and Medicine,
College of Physicians and Surgeons, Columbia
Univ., New York City.

Thin sections of HeLa cells infected with type 3
ARD (APC) virus and fixed in osmium tetroxide
revealed viral particles, frequently in character-
istic crystalline array (KJELLEN et al., Nature 175:
505, 1955; Harrorp et al., Bact. Proc. p. 64, 1955),
which appeared to differentiate from intranuclear
aggregates of chromatin. The particles exhibited
striking differences in density and averaged 60 mz
in diameter. The relatively constant space sepa-
rating them suggested the presence of some pe-
ripheral structure not clearly visualized, thereby
increasing the calculated diameter to approxi-
mately 70 my. A sharply defined central body
averaging 25 myz in diameter was observed in
numerous particles. Infected nuclei occasionally
contained canaliculi and lamellae arranged in
spiral form. Because these structures were not
consistently encountered and exhibited little
spacial relationship to adjacent virus, it appeared
unlikely that they were directly related to viral
development. Virus appeared to be released into
the cytoplasm by breakdown of nuclear mem-
branes. In the cytoplasm occasional crystals were
encountered but the virus was more often observed
as irregular aggregates or dispersed particles,
indicating that disintegration of the crystals had
taken place after release from the nucleus. The
intranuclear and intracytoplasmic forms of the
virus appeared to be identical in size and structure.
Although study of the micrographs suggested that
differentiation of the particles was confined to the
nucleus, development within the cytoplasm could
not be excluded.

1973. New tuberculostatic protein isolated
from bovine spleen. QuENTIN N. Myrvik.
Dept. of Microbiology, Univ. of Virginia, School
of Medicine, Charlottesville.

Aqueous extracts, prepared by acid (px 3.3) or
neutral extractions of freshly minced spleen, in-
hibit the virulent H37Rv (Human) and BCG
(bovine attenuated) strains of Mycobacterium
tuberculosis at a dilution of 1:48 based on the
weight of fresh spleen. In contrast, these prepa-
rations inhibit the virulent Ravenel strain (bo-

606 FEDERATION PROCEEDINGS

vine) at a dilution of 1:12. The active principle
has been isolated by alcoholic fractionation and
repeated isoelectric precipitations. Physicochemi-
cal studies on the active substance indicates that
it is nondialyzabie, negatively charged at px 7.0
and migrates as a single homogeneous band on
paper electrophoresis. In highly purified form it is
capable of inhibiting the H37Rv strain in a concen-
tration of about 30-40 ug/ml. The activity of this
new inhibitor correlates with the resistance of the
cow to active infection with the above strains of
tubercle bacilli. Thus, these data emphasize the
role of tissue inhibitors in native resistance to
tuberculosis.

1974. Antigen-antibody reactions in agar. III.
Rate of change of band migration with
antigen concentration. J. C. Nerr* anp E.
L. Becker. Immunology Div., Walter Reed Army
Inst. of Research, Washington, D. C.

In the Oudin serum agar technique, at suffi-
ciently low antigen (Ag) and high antibody (Ab)
concentrations Oudin showed empirically that an
equation of the following form holds: k = m log
Ag + m log R’ Ab (Eq. 1). In this equation, k is
the slope of the straight line obtained by plotting
x, the distance which the leading edge of the
precipitate moves in time, ¢, against t+. A theo-
retical derivation of (1) leads to the belief that m
might be proportional to D? where D is the diffu-
sion coefficient of the antigen. In corroboration of
this idea it was found that when varying dilutions
of egg albumin (Ea) were run against varying
concentrations of rabbit antiserum to Ea, and m
calculated for each antiserum dilution, there was
a tendency for m to decrease with increasing anti-
serum concentrations. Similarly, m tended to be
depressed when nonspecific protein such as normal
rabbit serum or bovine gamma globulin was
added to the serum agar layer. However, m could
be made effectively constant by multiplying it by
nt where » was the relative viscosity of the anti-
serum or antiserum protein mixture. The average
corrected value of m found for the Ea-anti Ea
system was 1.2; X 10 cm/min.!. The same
viscosity correction was shown to be applicable to
the human serum albumin (Hu.8.A.) rabbit anti-
Hu.S.A. system, and the average corrected value
was 1.04 X 10? cm/min.}. The ratio of mg, to
mau-s-a is 1.15 compared to 1.12 for the ratio of
the square roots of the diffusion coefficients of the
two antigens.

1975. Assays of plasmin, human activator-
enzyme and plasmin inhibitors. PHILIP
S. Norman (introduced by MEerriLu W. CHASE).
Rockefeller Inst. for Med. Research, New York
City.

A common method of assay is defined, based on

Volume 16

the digestion of 2% casein for 30 min. at 37°C and
measurement of trichloroacetic acid soluble prod-
ucts. For all 3 assay procedures the precipitate
from 0.5 ml of serum at pH 5.2 and reduced ionic
strength suffices as a source of enzyme. Animal
sera, €.g., guinea pig, can be assayed for total
plasmin by addition of a small amount of strep-
tokinase-activated human globulin to furnish
activator-enzyme. By this means, the amount of
plasmin in guinea pig serum is found to be about
double that present in human serum. Since guinea
pig serum is itself free of activator-enzyme, the
amount of this enzyme can be measured by its
ability to activate guinea pig plasminogen. Only
0.02 ml of human serum is required for the assay.
Purification of human plasminogen by Kline’s
method. (J. Biol. Chem. 204: 949, 1953) is found to
concentrate the activator-enzyme also. Spon-
taneous deterioration of plasmin at 37° presents a
difficulty in measuring its reaction with inhibitor,
However, plasmin is stable at 25° for at least 90
min. and inhibition can be tested accurately at
this temperature. When plasmin is allowed to
react to plasminogen-free serum proteins of the
guinea pig, two stages are noted, a rapidly partial
inhibition and a further slow progressive inhibi-
tion. The slow reaction is temperature dependent
and is practically nil at 0°C. These findings sug-
gest the presence of two serum inhibitors of
plasmin.

1976. Quantitative studies on specificity of
the Wassermann antibody. ABRAHAM G. OSLER
AND ELEANOR A. Knipp.* Dept. of Microbiology,
Johns Hopkins School of Hygiene and Public
Health, Baltimore, Md.

Specific aggregates formed by the interaction of
the Wassermann antibody with saline emulsions of
bovine cardiolipin, lecithin and cholesterol were
analyzed for antibody N by the quantitative
ninhydrin procedure. The method yielded precise
and reproducible results with human and rabbit
sera containing as little as 10-20 ug of antibody
N/ml. The reaction curves obtained with this
immune system could be described by the Heidel-
berger-Kendall equation for the quantitative
precipitin reaction. The data obtained in the
present study indicate that 0.015-0.03 ug of Was-
sermann antibody N suffice for a positive serologic
test in the laboratory diagnosis of syphilis.
Analyses were also carried out with human and

abbit Wassermann antibody and lipid soluble
antigens derived from human and plant tissues.

The bovine and human tissue antigens precipitate

essentially identical quantities of Wassermann

antibody from human sera. The results obtained
from quantitative complement fixation experi-
ments will also be discussed in relation to the prob-
lem of the specificity of the Wassermann antibody.

bot

ae eee ee eee ee ee ee ee ee i eee ee ee ee ee ee ee a a a ae

ume 1§

°C and
> prod-
ipitate
d ionic
Animal
r total
strep-
‘urnish
unt of
about
guinea
1e, the
by its
. Only
assay.
Kline’s
und to
Spon-
sents a
ibitor.
rast 90
ely at
ved to
of the
partial
inhibi-
ondent
rs sug-
ors of

ity of
OSLER
tology,
Public

‘ion of
ions of
| were
tative
precise
rabbit
tibody
h this
leidel-
tative
n the
- Was-
‘ologi¢
philis.
n and
oluble
issues.
pitate
rmann
tained
xperi-
prob-
ibody.

March 1956

1977. Mortality and body weight loss of
acutely and chronically fasted rats sub-
jected to surgical trauma. Morton D.
PAREIRA AND STANLEY LaN@ (introduced by
M. S. FLEIsSHER). Surgical Labs., Research Inst.,
Jewish Hosp. of Saint Louis, St. Louis, Mo.
Acutely and chronically fasted albino rats have

been subjected to surgical trauma of graded in-
tensities as part of a larger study designed to
characterize a purported state of enhanced surgi-
cal risk due to under- or malnutrition. The cri-
terion used to evaluate the effects of surgical
trauma applied to underfed animals were daily
body weight loss and survival times. These indices
demonstrated gross differences between control
fasting animals and fasting rats whose hormonal
environment had been altered. Fasting or chroni-
cally underfed rats subjected to surgical trauma,
however, failed to show any differences in either
daily body weight loss or survival time when com-
pared to their controls. Surgical trauma consisted
of laparotomy and exteriorization of the abdomi-
nal viscera which were wrapped in a dry, sterile
sponge for varying lengths of time. The longest
period of exposure was 1 hr. The trauma was
applied on different days of fasting.

1978. Hemopoietic response in mice injected
with pertussis vaccine. I. A. PARFENTJEV
AND E. E. Manvueuipis.* Depts. of Microbiology
and Pathology, Yale Univ. School of Medicine,
New Haven, Conn.

Mice immunized with pertussis vaccine de-
veloped resistance to this organism, but at the
same time acquired nonspecific susceptibility to
infection caused by a number of unrelated agents
such as Proteus, Pasturella, Burcella. Further-
more, these mice became more susceptible to
Shigella endotoxin and to influenza virus. The
impairment of the capacity of antibodies to
neutralize the infectious agent in hypersensitive
mice was reported earlier (PARFENTJEV ef al.,
1947; PARFENTJEV, 1955). It is difficult to interpret
these findings solely on the basis of antibody
action. In studies of white blood cells we have
found that the injection of pertussis vaccine causes
a marked leucocytosis in mice (CFW strain) and
that the elevated level of white blood cells persists
for at least 2 wk.; this leucocytosis is due chiefly
to an increase in the granulocytes. However, in the
first few days after injection of vaccine, lympho-
cytes also are moderately increased in number, but
soon decrease while the elevated level of the
granulocytes persists. The spleens of sensitized
mice are approximately twice as heavy as those of
normal mice. The white pulp in such spleens is
reduced in size, but the red pulp is considerably
increased; this could be responsible for the in-
crease in weight. We have considered three possi-

AMERICAN ASSOCIATION OF IMMUNOLOGISTS

607

bilities for non-specific susceptibility in mice sensi-
tized by pertussis vaccine: /) impairment of
natural antibodies in the state of hypersensitivity;
2) enlargement of red pulp indicating an increase
in filtering capacity of the spleen, possibly in con-
nection with elimination of dying granulocytes,
which might interfere with the elimination of
bacteria from the body; 3) a reduction of the white
pulp of the lymphatic part of the spleen.

1979. Antagonists of murine encephalo-
myelitis virus, in vitro. HARoLD E. PEARsoN,
Dorotoy L. LacerBorG* AND Donatp W.
VisseR.* Depts. of Public Health and Bio-
chemistry and Nutrition, Univ. of Southern
California School of Medicine and Lab. Div.,
Los Angeles County Hosp., Los Angeles.
Previously several compounds were found to in-

hibit the propagation of Theiler’s GD VII strain

of mouse encephalomyelitis virus in flask tissue
cultures of minced, newborn mouse brain. In this
study virus inhibition occurred in the presence of
certain substituted pyrimidine nucleosides,
5-bromo-, 5-chloro- and 5-hydroxycytidine, deoxy-
adenosine, cytidine and guanosine. Deoxycytidylic
acid, deoxyuridine, 5-bromodieoxyuridine and
5-hydroxydeoxyuridine were not inhibitory; 5-hy-
droxyuridine inhibited, and this inhibition was
partially reversed by uridine-5’phosphate. Pyrimi-
dine derivatives which inhibited virus included
2-amino-4-methylpyrimidine, and 4,6-diamino-2-
thiopyrimidine but not 2,4-dichloropyrimidine or
2,4-dichloro-6-methypyrimidine; 5-bromouracil,
5-methyl-2-thiouracil and 6-methyl-2-thiouracil
were slightly inhibitory, as was 2-thiouracil in
relatively high concentrations. Propylthiouracil
inhibited, as did theophylline. The action of the
former was not reversed by uracil. Several thio-
semicarbazones found elsewhere (THOMPSON e¢ al.
J. Immunol. 70: 299, 1953) to inhibit vaccinia
virus were tested against Theiler’s virus. Butane-
2,3-dione-2-methoxime-3-thiosemicarbazone was
inhibitory. Some other inhibitory compounds were
pipecolic acid (slightly active), quinine dihydro-
chloride and chlorpromazine (Thorazine) which

were highly active and a less active drug 2,2-di-

ethyl-1,3-propanediol dicarbamate (Metuchen).

Polymyxin inhibited virus, in vitro, but did not

protect mice from fatal infection.

1980. Binding of S* complement. NATHAN
PEenN,* MuTAHHAR YENSON* AND FELIX HavuRo-
witz. Dept. of Chemistry, Indiana Univ., Bloom-
ington.

The binding of complement by antigen-antibody
precipitates was studied with guinea pig sera
labeled by feeding S**-amino acids. S** guinea pig
sera, heat-inactivated or decomplemented by a
separate antigen-antibody system, served as con-

608

trols. Appreciable adsorption of activity from the
controls was observed. The excess activity bound
by the test system from active guinea pig sera
over that bound from controls was 0.4-0.7% of the
total serum activity. The counts adsorbed at 0°
and 37° by antigen-antibody precipitates were
found to be approximately equal. It is known that
the major reaction to 0° is the binding of C’l
(Lepow AND PiLuemER, J. Immunol. 75: 63, 1955).
It therefore appears that, within the limits of
experimental error, there is no significant perma-
nent combination of other complement compo-
nents with antigen-antibody aggregates after the
initial adsorption of C’l. (Support of this work by
the Public Health Service, the Natl. Science
Fndn. and U. 8. Atomic Energy Commission and
the Office of Naval Research is gratefully acknowl-
edged.)

1981. Effects of salicylic acid on aerobic
respiration of rat brain preparations.
RaA.PH PENNIALL (introduced by K. L. Burpon).
Dept. of Biochemistry, Baylor Univ. College of
Medicine, Houston, Texas.

Salicylic acid was studied for its effects on the
oxidation of pyruvate and succinate by homoge-
nates and mitochondria derived from rat brain.
Oxidation of pyruvate was studied by means of
10% isotonic KCl homogenates. Both substrates
were used in the work with mitochondria. The
drug had no distinct effect on the oxygen uptake
of pyruvate oxidation with either homogenates or
mitochondria. However, at 10-4 m concentration
the drug exhibited significant inhibition of the up-
take of inorganic phosphate accompanying the
oxidation of pyruvate. This uncoupling action was
observed with both homogenates and mito-
chondria. Drug effects upon phosphorylation in
homogenates were greater than those observed
with mitochondria. At 2 X 10-3 m concentration,
phosphate uptake was nullified with homogenates
whereas with mitochondria phosphorylation was
only inhibited 50%. This uncoupling action was
found to be reversible by the criteria of Acker-
mann and Potter (Proc. Soc. Exper. Biol. & Med.
72: 1, 1949}. Further work on the mitochondrial
oxidation of succinate gave similar evidence of the
drug’s uncoupling action. The effects of the drug
on the oxidation of other Krebs’ cycle inter-
mediates will also be discussed. (Supported in
part by a grant from the American Med. Assn.)

1982. Physical binding of insulin by gamma
globulins from insulin-resistant subjects.
THEODORE PETERS,* Betton A. BurRrows*
AND Francis C. LowE.u. Radioisotope Service,
Boston VA Hosp., Robert Dawson Evans Me-
morial, Massachusetts Memorial Hosp., and
Dept. of Medicine, Boston Univ. School of Medi-
cine, Boston, Mass.

FEDERATION PROCEEDINGS

Volume 16

Insulin was iodinated to the extent of 3 ue of
I'3!/ug and about 1 atom of iodine/molecule. The
I'3!_jnsulin, when added zn vitro to sera of normal
persons or of unselected patients with diabetes
mellitus, did not migrate upon paper electro-
phoresis (Whatman 3MM paper, pH 8.6, »/2 0.05,
5 v/em, 16 hr.), but remained adsorbed to the
paper at the starting point. When added to sera of
persons of known insulin resistance, however, the
I'5!_insulin migrated with the middle or leading
portion of the gamma globulin fraction. The
effect is attributed to binding by nonprecipitating
antibodies, the electrophoretic mobility of the
complex differing only slightly if at all from that
of gamma globulin. By adding progressively larger
amounts of insulin, in most cases the concentration
of insulin needed to ‘saturate’ a given serum could
be estimated. Seventeen sera from 4 insulin-
resistant diabetic patients and 1 resistant rabbit
were shown to bind insulin in amounts from about
0.05 ug to more than 20 ng/ml. Values correlated
well with the clinical behavior and results of
mouse tests for neutralization of insulin by the
same sera. Absence of a cross-reaction between
human and beef insulin was demonstrated in the
serum of one patient. The above technique should
prove of general value as a sensitive test for the
presence of nonprecipitating antibodies.

1983. Fixation of complement by sensitized
erythrocytes. O. J. PLescta AND K. AMIRAIAN
(introduced by M. HrmpELBERGER). Inst. of
Microbiology, Rutgers Univ., State Univ. of New
Jersey, New Brunswick.

Because immune hemolysis consists of a se-
quence of steps involving the fixation of compo-
nents of complement (C’), the thermodynamics of
the over-all reaction can be explained in terms of
specific characteristics of the individual steps.
Fixation of components of guinea pig, human and
pig C’ by sensitized erythrocytes in excess was
studied as a function of concentration of sensi-
tized cells and C’, temperature, time and relative
concentrations of the components of C’. The fol-
lowing results were obtained: with guinea pig C’,
the only one tested in this respect, 1) fixation of
C’1, C’2 and C’4 is essentially complete for tem-
peratures ranging from 0°C to 37°C, although
considerable time is needed at 0°C; 2) once fixed,
C’l, C’2 and C’4 are not measurably dissociated
either at 37°C or 0°C; 3) with reagents from pig
serum, C’l and C’4 were shown to fix independ-
ently of each other as were also C’l and C’2;
4) the distribution of components of C’ fixed by
sensitized cells depends on the composition of C’
and hence on the source of C’. The implications of
these findings with respect to the mechanism of
immune hemolysis and the fixation of C’ by im-
mune specific precipitates will be discussed.

ume 16

3 uc of
e. The
10rmal
abetes
lectro-
2 0.05,
to the
sera of
er, the
eading
.. The
itating
of the
m that
larger
ration
1 could
nsulin-
rabbit
about
elated
ts of
by the
tween
in the
should
or the

itized
'RAIAN
st. of
if New

a se-
ompo-
nics of
rms of
steps.
in and
33 was
sensi-
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ne fol-
ig C’,
ion of
r tem-
hough
fixed,
ciated
m pig
»pend-
1 C2;
ed by
of C’
ons of
ism of
yy im-

March 1956

1984. Antibody-comibining properties of bac-
terial flagella. Kurvenat Reap, MERWwIN
Moskowitz AND Henry Korrier (introduced
by F. JoserpH Murray). Labs. of Bacteriology,
Dept. of Biological Sciences, Purdue Univ., West
Lafayette, Ind.

The antibody-combining properties of purified
flagella (from Proteus vulgaris) and their ‘de-
polymerized’ products were determined by quanti-
tative precipitin tests. The flagella were isolated
and purified by differential centrifugation, after
they had been shaken off the cell bodies. The
precipitin reaction apparently is influenced by the
fibrous nature of the flagella. With an increase in
the amount of flagella added to the antiserum
there is a striking increase in the viscosity of the
mixture. The initial portion of the precipitin curve
is similar to that of other protein-antiprotein
systems, but the final portion of the curve deviates
from that obtained with purified soluble proteins.
The antibody content of the antiserum studied
was 0.5 mg/N/ml. At the point of maximum
precipitation of antibody, the ratio of antibody N
to flagella N is 1. When subjected to heat (60°C,
30 min.) or acid (pH 2, 25°C, 30 min.), flagella lose
their characteristic structure, as judged by
electron microscopy and viscosity measurements,
but the products formed from such treatments
still precipitate with antibodies against whole
flagella.

1985. Sequence of morphological changes in
poliovirus, infected cells in culture, corre-
lated with growth cycle of the virus. Maapa-
LENA Rerssic,* Davin HowrEs* AND JOSEPH L.
Metnicx. Sect. of Preventive Medicine, Yale
Univ., New Haven, Conn.

A study was made of the morphological changes
observed in cultures of monkey kidney cells at
different times after poliovirus inoculation. Alter-
ation of the chromatin pattern of the nucleus and
intranuclear inclusions were seen as early as 4 hr.
after virus inoculation. Later, wrinkling and
shrinkage of the nucleus and eosinophilic cyto-
plasmic masses appeared. The rounded pycnotic
cell, customarily used as an index of the cyto-
pathic response, was found only during the very
late stage of the infective process. Based on these
changes, infected cells could be classified into six
different types. Differential cell counts were made
on stained cultures at varying periods after inocu-
lation, and the stage of cytopathic degeneration
was correlated with the appearance of newly
formed virus in the cells and in the culture fluid.
A delay in the appearance of the morphological
changes was accompanied by a corresponding
delay in virus production. The virus-induced
morphological changes exhibited a specificity
distinct from the classical pycnosis of autolytic

—r
ave tae

AMERICAN ASSOCIATION OF IMMUNOLOGISTS

609

degeneration. Cultures of monkey kidney epi-
thelium which had spontaneously undergone the
foamy type of degeneration and multinucleated
celi formation described by Enders and Peebles
and by Rustigian, were superinfected with polio-
virus. Within a day after virus addition, the multi-
nucleated giant cells underwent the cytopathic
changes described above for poliovirus infected’
cells. Both normal nuclei and nuclei showing
poliovirus induced changes were found in the same
multinucleated cell.

1986. Electron microscopic studies of struc-
ture and development of influenza virus.
Harry M. Rose, CouncrtMaAN MorGan* AND
Dan H. Moors.* Depts. of Microbiology and
Medicine, College of Physicians and Surgeons,
Columbia Univ., New York City.

This sections of chorioallantoic membranes
were fixed in osmium tetroxide 4—44 hr. after in-
fection. Rod-like and spherical forms of the virus
were seen. Although the rods had no visible in-
ternal structure, the spheres possessed a discrete,
spherical, central body averaging 20 my in di-
ameter. Both forms exhibited a poorly demarcated
peripheral zone surrounding a sharply defined,
dense membrane. A relatively constant space
(20-25 mz) separated the viral particles. Assuming
that such spacing reflected the presence of struc-
ture not defined by the electron microscope, the
diameter of a majority of the rods could be calcu-
lated to be 50-60 mu, whereas the spheres averaged
70 mu. Little evidence was obtained to support the
hypothesis that the spheres are formed by seg-
mentation of the rods. Segmentation is believed to
reflect an artifact of drying. Rods and spheres
appeared to develop separately at the cell surface.
Particles considered to be virus were encountered
only at the surface of the host cell and no specific
changes were observed either in the nuclei or
cytoplasm. This study raises the question whether
two forms of the same virus exhibiting such
marked differences in shape and internal structure
are both capable of initiating infection.

1987. Production of specific rabbit thyroid
antibodies in the rabbit. Nort R. Rose* anp
Ernest Wiressky. Dept. of Bacteriology and
Immunology, School of Medicine, Univ. of Buf-
falo, Buffalo, N. Y.

Rabbits were injected with pooled rabbit thy-
roid crude extract plus Freund adjuvants intra-
dermally into the footpads. Most of the animals
developed antibodies which reacted with rabbit
thyroid extract in complement fixation, precipi-
tation, and Boyden’s tanned cell hemagglutina-
tion tests. The antisera were found to possess the
following serological characteristics, indicating
that they are thyroid-specific rather than group-
specific in nature: /) the antisera reacted with a

610

large number of individual rabbit thyroid extracts;
2) thyroidectomized rabbits injected with an
extract of their own thyroid glands produced
thyroid antibodies; 3) the antisera did not react
with extracts of other rabbit organs; 4) cross-
reactions with thyroid extracts of certain other
species were obtained; 5) the antisera reacted with
the extracts of the rabbits’ own thyroid glands. A
small series of rabbits injected intravenously with
relatively large amounts of rabbit thyroid extract
failed to produce any demonstrable antibodies,
but a small proportion of a series of rabbits in-
jected intradermally with the antigen-adjuvant
mixture omitting the acid-fast bacilli did form
thyroid antibodies. The antibody is stable at
56°C for 30 min., but destroyed when heated to
80°C for 10 min. In starch-supported electro-
phoresis, it migrates with the gamma globulins.
Immunized rabbits show histological and sero-
logical evidence of thyroid damage. The antibodies
apparently fulfill the criteria of autoantibodies.

1988. Mutual inhibition of streptococcus
mitis and Streptococcus pyogenes group A.
THEODOR RoSEBURY, JACQUELYN R. ZEITINGER*
AND JosePH J. Mocas.* Dept. of Bacteriology,
Washington Univ. School of Dentistry, St. Louis,
Mo.

The previous observation of mutual inhibition
on cross-titration plates of S. mitis and S. pyogenes
(J. Bact. 67: 135, 1954) has been studied further.
In a series of experiments utilizing 1 strain of S.
mitis and 2 strains of S. pyogenes group A on
rabbit blood agar aerobically and anaerobically, at
both 10-fold and ~/10-fold dilution intervals,
undertaken as part of a statistical study of the
cross-titration method (details of which will be re-
ported elsewhere), mutual inhibition appeared
regularly. Two separate fronts of inhibition could
be plotted on each cross-titration (log-log) grid,
both being satisfactorily characterized as linear.
Cross-titrations with 9 additional strains of S.
mitis and with 10 additional strains of S. pyogenes
group A all showed characteristic mutual in-
hibition. Mixed cultures of the 2 species in differ-
ent inoculum concentration ratios in blood-thio-
glycollate broth have shown clear inhibition of
S. pyogenes in growth curves (pour plate counts)
and by mouse inoculation. Inhibition of S. mitis
has not as yet been clearly demonstrated in broth.
Further attempts to do so, and to provide clues to
the inhibitory mechanism(s), are under way. Both
inhibitions seem to require close microbic associ-
ation; their occurrence anaerobically on blood
agar indicates that H.O2 is not involved. These
and other data suggest a role for nonhemolytic
streptococci in natural resistance, e.g., in the
human mouth and throat, exerted as part of the
pattern of microbic ecology in these areas.

FEDERATION PROCEEDINGS

Volume 1§

1989. Mechanism of induced refractoriness of
mice to streptolysin O. RoBERT ROWEN* AnD
ALAN W. BERNHEIMER. Microbiology Dept.,
New York Univ. College of Medicine, New York
City.

Mice injected intravenously with one or more
doses of streptolysin O rapidly develop refractori-
ness to a subsequently administered lethal dose.
The plasma of such mice is about 10 times as
effective as normal mouse plasma in inhibiting in
vitro the hemolytic action of streptolysin O. The
inhibitory activity of plasma of refractory mice
a) is nondialyzable, b) separates with albumin on
ammonium sulfate fractionation, c) is precipitated
by cold methanol, d) is increased by freeze-drying
and heating to 56° and e) is largely removed or
destroyed by ether extraction at —70°. These and
other properties suggest that the inhibitor is
lipoprotein. The inhibitory substance(s) is quanti-
tatively floated by ultracentrifugation of re-
fractory mouse plasma of adjusted density 1.060
gm/ml (24°). Starch electrophoresis of ultracen-
trifugally separated lipoprotein yields two zones
of inhibition: one with a mobility in the region of
a-] lipoprotein and one in the region of 6-globulin.
It is concluded that the induced refractoriness is
due to the presence in blood of certain lipoproteins
in concentrations greater than in blood of normal
mice. (Supported in part by the Arthritis and
Rheumatism Fndn. and the Life Insurance Med,
Research Fund.)

1990. Measles in humans and in monkeys:
report of isolation from cynomolgus mon-
keys of an agent immunologically related to
human measles virus. GISELA RUCKLE (in-
troduced by Jonas E. Sak). Virus Research
Lab., School of Medicine, Univ. of Pittsburgh,
Pittsburgh, Pa.

Following the report by Enders and Peebles,
viral agents, transmissible in cultures of either
trypsinized monkey or human kidney tissue, have
been isolated from blood and/or throat washings
obtained from 6 patients with typical measles.
We have observed that continued passage in
monkey kidney cultures sometimes results in loss
of these agents. It now appears that rhesus kidney
cell cultures do not sustain measles virus as well
as cultures of cynomolgus kidney tissue. Re-isola-
tion in human kidney cultures from original ma-
terial was successful after 6 months storage at
—70°C. Cultures of human amniotic membrane are
now being used for passage. During these studies
two different types of transmissible agents have
been found in uninoculated monkey kidney tissue
cultures. One resembles the so-called ‘foamy
virus’ and the other produces intranuclear inclu-
sions that are indistinguishable from that pro-
duced by the agent obtained from human measles.

~~ oo.w «©

lume 16

ness of
oN * AND

Dept.,
w York

yr more
ractori-
il dose.
mes as
iting in
O. The
‘y mice
min on
pitated
-drying
ved or
ese and
itor is
quanti-
of re-
y 1.060
tracen-
) zones
gion of
obulin.
iness is
roteins
normal
is and
e Med.

ankeys:

mon-
ted to
E (in-
esearch
sburgh,

eebles,
either
1, have
shings
easles.
ge in
in loss
<idney
is well
-isola-
al ma-
age at
ne are
tudies
; have
tissue
foamy
inclu-
{ pro-
2asles.

March 1956

Each of these agents has been encountered on six
different occasions and under circumstances where
the possibility of contamination with human
measles virus could be excluded. Two cynomolgus
monkeys that had no measles antibody upon
arrival in the laboratory were found to have had
such antibody 3 months later. The evidence sug-
gests acquisition of infection from other monkeys,
since human measles virus had not been deliber-
ately introduced into the monkey colony experi-
mentally. Cross neutralization tests reveal that
the ‘foamy virus’ is different from the monkey
intranuclear inclusion agent and that the latter is
immunologically indistinguishable from human
measles virus.

1991. Bile acid content of serum and urine in
hepatic disease. DaNriEL RUDMAN* AND
Forrest E. Kenpauu. Columbia Univ. Research
Div., Goldwater Memorial Hosp. and Dept. of
Medicine, Columbia Univ., New York City.
Chromatographic and spectroscopic methods

for the separation and identification of bile acids

have been applied to the study of the bile acid
content of serum and urine. No bile acid was de-
tected in serum or urine of individuals without
liver disease. In 7 patients with Laennec’s cir-
rhosis, varying amounts of dihydroxy bile acid
(0.7-9.0 mg %) were present in serum; no bile acid
was found in urine. In 4 cases of obstructive liver
disease, both dihydroxy bile acid (2.5-7.0 mg %)
and cholic acid (1.4-11.0 mg %) were present in
serum, and also in urine (9-35 mg daily for each
bile acid). The bile acids in the serum and urine
of patients with obstructive hepatic disease were
of the conjugated type; the nature of the serum
dihydroxy bile acid in Laennec’s cirrhosis is under
study. The bile acids in serum, whether free or
conjugated, are bound by the serum albumin. The
degree of binding is greatest with the mono-
hydroxy and least with the trihydroxy bile acids.

Evidence will be presented for the existence of an

electrostatic bond between the carboxyl radical of

the bile acid and the positively charged side chains
of the albumin molecule.

1992. Effect of prolonged formaldehyde treat-
ment on antigenic activity of different
strains of poliomyelitis viruses. Jonas E.
Satk, Byron L. Bennett,* L. J. Lewis* anp
Francis Yurocuko.* Virus Research Lab.,
School of Medicine, Univ. of Pittsburgh, Pitts-
burgh, Pa.

In extension of numerous studies, unpublished
as yet, on destruction of poliomyelitis virus in-
fectivity by formaldehyde, further work has been
carried out on the relative stability of the anti-
genic component after infectivity is no longer
demonstrable. In the studies to be reported, the
reaction temperature employed was 36.5°C and

AMERICAN ASSOCIATION OF IMMUNOLOGISTS

611

acidity adjusted to pu 7.0. Formaldehyde content
was varied in 2-fold steps, using formalin in final
concentration of 1:500 to 1:4000, and also at
1:6000. The rate of loss of virus infectivity in-
creased with increasing concentration of formalin;
infectivity in 0.5-ml samples was no longer demon-
strable after less than 1 day of treatment, with the
greatest concentration, and after about 3 days
with the lowest concentration of formalin. How-
ever, antigenic activity, as measured both in terms
of antibody inducing capacity for animals, and in
terms of antibody combining capacity (Krech),
was still present long after disappearance of in-
fectivity. In this respect there were differences
among strains in the stability of their antigenic
moieties while the infective components seemed to
be destroyed at rates that were indistinguishable.
For example, the Saukett strain of type 3 virus,
in the presence of 1:4000 formalin, for 40 days at
36.5°C, still retained antigenic activity, whereas
the Mahoney strain of type 1 virus lost activity
gradually until after 30 days of such treatment,
after which activity was sharply reduced by the
40th day. With the greater concentrations of
formalin, corresponding but more rapid effects
were observed.

1993. Sensitivity response of tissue cultures
to various types of inactivated poliomyelitis
virus. RayMonp W. SarBer, W. B. Brarp-
MORE AND A. E. Hook (introduced by I. Wi1-
LIAM McLEAN, JR.). Research Labs., Parke,
Davis and Co., Detroit, Mich.

An extensive series of safety tests have been
performed in the course of the development of
both a method for inactivation of poliomyelitis
virus and a reliable method for testing for non-
viability of the virus. The general method for
testing has been the combining of equal parts of
virus suspension and culture medium in bottles of
trypsinized monkey kidney fibroblasts with a
change of medium and subculturing at weekly
intervals. Throughout a prolonged testing pro-
gram on various types of formalin inactivated
poliomyelitis virus suspensions, the test procedure
has been complicated by nonspecific sloughing and
cellular degeneration, making it necessary to
regularly replace bottles in order to complete the
tests. The use of .5% per cent calf serum improved
the stability of the cell sheath and permitted in-
creasing the holding of bottles from 14 days to a
total of 28 days with indications of increased sensi-
tivity, but did not eliminate nonspecific sloughing
and cellular degeneration. However, in a similar
number of 28-day tests on a series of ultraviolet
irradiated poliomyelitis virus suspensions and a
somewhat lesser number of 14-day tests, no tests
failed to go to completion on the original bottles.
The test tissues in the irradiated series were

612

maintained in better condition than in the non-
irradiated series, with a bottle loss of less than
1%, usually occurring during the 4th week of the
test.

1994. Oxidative decarboxylation of malate by
Ascaris lumbricoides. Howarp J. Saz (intro-
duced by M. Tacer). Dept. of Pharmacology,
Louisiana State Univ., School of Medicine, New
Oricans.

Succinate is present in high concentration in the
perienteric fluid of Ascaris. No detectable quanti-
ties of other dicarboxylic acids can be found
despite the occurrence of a predominantly anaer-
obic metabolism and of a potent succinic de-
hydrogenase (BUEDING AND Farrow, unpublished
observations). Homogenates of Ascaris muscle
catalyzed the anaerobic decarboxylation of
fumarate and malate. The products were lactate
and carbon dioxide. The rate of fumarate de-
carboxylation could account for the absence of di-
carboxylic acids other than succinate from the
perienteric fluid. Purified fractions were obtained
which were free of fumarase activity and required
1-malate as the substrate. The decarboxylation of
malate required Mn** and DPN. TPN could re-
place DPN only partially. In contrast to the
‘malic enzymes’ of pigeon liver (Ocuoa et al. J.
Biol. Chem. 167: 871, 1947) and of Lactobacillus
(Korkss et al. J. Biol. Chem. 176: 463, 1948),
purified Ascaris preparations were free of demon-
strable lactic dehydrogenase and did not catalyze
the decarboxylation of oxalacetate (between pH
5 to 8; with or without added ATP). Maximal ac-
tivity was observed upon the addition of lactic
dehydrogenase. In the absence of added lactic de-
hydrogenase, stoichiometric amounts of pyruvate,
DPNH and carbon dioxide were formed from
malate. In contrast to Ascaris, no ‘malic enzyme’
could be detected in mammalian muscle (OcHoa.
Physiol. Rev. 31: 56, 1951). The incorporation of
isotopic carbon dioxide into malate is being in-
vestigated. (Supported by PHS grant # E-668).

1995. Microscopic observations of living
rickettsiae. M. ScHAECHTER AND F. M. Boze-
MAN (introduced by E. B. Jackson). Walter
Reed Army Med. Ctr., Washington, D. C.
Ricketisia tsutsugamushi was grown in tissue

cultures of normal rat fibroblasts (strain 14pf) and

observed by phase microscopy. Explants of fibro-
blasts were placed on coverglasses and rotated in
roller tubes containing nutrient fluid. After 24 hr.
the resulting monolayers of cells were infected
with partially purified suspensions of rickettsiae.

Chambers containing infected cultures were made

at suitable intervals by inverting a coverglass on a

microscope slide and sealing the edges with a

paraffin-beeswax mixture. These chamber cultures

were observed and photographed for 8-hr. periods

FEDERATION PROCEEDINGS

Volume 16

while held at 33-38°C in a stage incubator. Intra-
cellular rickettsiae were seen in stained prepa-
rations of culture cells shortly after infection but
could not be recognized with certainty in living
cells until several days later, at which time they
occurred as clusters near the nucleus. Rickettsiae
constantly changed position in the cytoplasm;
within a few hours clumps of organisms dispersed
and reaggregated. Occasionally, rickettsiae previ-
ously seen within cytoplasmic peduncles were left
outside the cell when the peduncle retracted. Fur-
thermore, extracellular rickettsiae sometimes
entered a cell when a cytoplasmic protrusion or the
cell border flowed over the organisms. No single
organism has yet been observed undergoing a
complete cycle of division.

1996. Sero-survey of arthropod-borne virus
infections of Iran and Afghanistan resi-
dents. J. R. Scumipt, E. L. BuescHer anp D,
C. GaspusEK (introduced by J. A. Morais),
Waltdr Reed Army Med. Ctr., Washington, D. C.,
and Univ. of Maryland Med. School, Baltimore.
In investigating the occurrence, distribution

and prevalence of certain infectious diseases in
the Middle East, sera from indigenous adult
residents of seven Iranian and four Afghan com-
munities located in different geographic and
ecologic settings were tested for antibodies against
Casals’ Group B arthropod-borne viruses. For
initial screening, the hemagglutination-inhibition
(HI) test was employed with Japanese encephalitis
E(3) and type 1 dengue antigens. A high incidence
(35-100%) of positive reactors occurred in western
Iran, Teheran, the eastern Caspian coastal regions
of Iran and in Afghanistan south of the Hindu
Kush mountain range. On the other hand, the inci-
dence of Group B HI antibody was low (under
5%) in residents of the western Caspian coastal
region of Iran and of Afghan villages north of the
Hindu Kush. Neutralizing antibodies against
West Nile (WN), JE and Ntaya (NT) viruses were
each demonstrable in practically all selected sera
containing Group B HI antibody but neutralizing
antibodies against Russian spring-summer en-
cephalitis, types 1 and 2 dengue, Ilheus, Semliki
Forest and Zika viruses were absent. The specific
agent responsible for JE, WN and NT neutralizing
antibodies in selected sera could not be incrimi-
nated on results of virus-dilution or serum-dilution
tests. Limitations in the application of serological
survey data to epidemiologic studies of arthropod-
borne virus diseases will be discussed.

1997. Effect of uremia on rate of urea synthe-
sis by rat liver slices. ALviN L. SELLERS,*
JosEPH Katz,* SHELDON RoOSENFELD* AND
Jesste Marmorston. Inst. for Med. Research,
Cedars of Lebanon Hosp., Los Angeles, Calif.
There is a disagreement in the literature con-

a owe — i ee en - e

—
| ~4

ti
n

lume 16

_ Intra-
prepa-
ion but
| living
1e they
ettsiae
plasm;
spersed
) previ-
ere left
d. Fur-
.etimes
1 or the
single
oing a

virus
| resi-
AND D,
)RRIS),
D.C.,
nore.
bution
ses in

adult
n com-
c and
igainst
s. For
ibition
halitis
idence
restern
egions
Hindu
e inci-
(under
-oastal
of the
gainst
'S were
d sera
alizing
or en-
emliki
pecific
alizing
crimi-
lution
logical
ropod-

nthe-
LERS,*
* AND
search,
if.

e con-

March 1956

cerning the effect of uremia on the rate of urea
formation in intact animals. To investigate this
further, we have studied the incorporation of
CO. into urea by liver slices taken from control
and uremic rats. Animals were made uremic by }
or total nephrectomy. Liver slices were incubated
by C'* bicarbonate in serum from normal and
uremic rats and in bicarbonate and phosphate
buffers. It was found that liver slices from uremic
rats incorporate from 1} to 4 times more CO,
into urea than controls. This occurred in the four
incubation media mentioned above and under a
variety of dietary conditions. The incorporation of
CO, into compounds other than urea was essen-
tially the same in liver slices from uremic and
normal rats.

1998. Growth characteristics of certain myco-
bacteria in HeLa cells. CHARLES C. SHEPARD.
Virus and Rickettsial Labs., Communicable Dis-
ease Ctr., PHS, Montgomery, Ala.

When certain horse sera are employed in tissue
culture media the number of tubercle bacilli taken
into HeLa cells is greatly increased. Use has been
made of this phenomenon to introduce various
mycobacteria into HeLa cells, so that their growth
in these cells of human origin could be observed.
Medium containing horse serum was used only for
the Ist day, and human serum thereafter. Among
the tubercle bacilli there was, in general, a positive
correlation between the pathogenicity for humans
and the growth rate in HeLa cells. The standard
‘pathogenic’ human and bovine strains grew well,
and within 3 days had occupied much of the cyto-
plasmic volume. Such strains as H37Ra and BCG
grew more slowly and in characteristic patterns.
The formation of long ‘cords’ was a distinctive
feature of freshly isolated strains and some old
‘pathogenic’ laboratory strains such as H37Rv.
Within cells infected with these strains, strands
looped about the nuclei were often seen. However,
some strains which had lost the ability to form
intracytoplasmic cords still grew well in the cells.
The intracellular growth of several INH-resistant
strains was not greatly different from INH-
susceptible strains. Mycobacterium balnet, which
has a lower temperature optimum than 37°C and
which is capable of producing lesions in human
skin, grew well in HeLa cells and better at 35°C
than at 37°. Tubercle bacilli grew more rapidly
at 37°C than at 35°.

1999. Preparation of hog _ thyroglobulin.
Sipney SwHuLMAN. (introduced by ERNEsT
Wiressky). Dept. of Bacteriology and Immunol-
ogy, School of Medicine, Univ. of Buffalo, N. Y.
Ultracentrifugal examination of crude saline

extracts of hog thyroids shows that 80% of the

sedimenting material is a component with sedi-
mentation rate of 19.3-1.82 ¢ (in Svedberg units

AMERICAN ASSOCIATION OF IMMUNOLOGISTS

613

and g/dl). This has been identified as thyro-
globulin. By a simple fractionation with am-
monium sulfate, a product containing about 95%
of this component is obtained. The saturated
ammonium sulfate that is employed is adjusted
with a little ammonium hydroxide to pu 7-8,
and with a little water to 4.00 m. The purified
thyroglobulin is found in the fraction of the thy-
roid extract precipitating between 1.60 m and
1.70 m of ammonium sulfate. Serological studies
employing an antiserum to the crude extract have
shown that the purified thyroglobulin retains the
high degree of organ-specific activity characteris-
tic of the original extract. By increasing the pro-
tein concentration of the supernatant, followed
by adjusting the salt level to give 2.50 m to 3.00 m,
a fraction is obtained which is a highly purified
preparation of one of the minor components, sedi-
menting at 4.0 Svedberg units. Preliminary studies
employing isoelectric precipitation and cold
ethanol precipitation have also been made.

2000. Influence of route of injection of teta-
nus toxoid on immune response in X-ir-
radiated mice. Myron S. SILVERMAN AND
Paut H. Cun (introduced by S. S. Etsere).
Biological and Med. Sciences Div., U. S. Naval
Radiological Defense Lab., San Francisco, Calif.
Studies previously reported from this laboratory

have shown that the immune response of x-ir-

radiated mice to tetanus toxoid injected sub-
cutaneously is dependent upon the time elapsing
between the injection of the toxoid and exposure
to x-ray, and upon the dose of x-ray to which the
mice were exposed. Regardless of whether ir-
radiation preceded or followed the injection of
antigen, the immune response, as measured by
challenge with 10 mup of tetanus toxin, was found
to be delayed, providing the dose of x-rays was
sufficiently high. Further studies have shown that
the immune response is also dependent on the
route of injection of the tetanus toxoid. Both
irradiated and nonirradiated mice injected intra-
venously with alum precipitated tetanus toxoid
developed sufficient immunity to protect against
10 MLD of tetanus toxin more slowly than did mice
injected subcutaneously. Nonirradiated animals
injected subcutaneously were able to survive the
challenge dose 2 wk. after immunization, whereas
intravenously injected mice required 5 wk. to
develop 100% protection. If the mice were exposed
to sublethal or low lethal doses (380-490 r) of whole
body x-irradiation, those animals injected with
toxoid subcutaneously showed a delayed response,
but by 5-6 wk. after immunization all were pro-
tected. Only a small percentage of the animals
given the toxoid intravenously were able to sur-
vive the challenge dose even 6 wk. after immuniza-
tion. Again the time necessary for the develop-

614

ment of the immune response was dependent on
the relationship of the time of injection to the time
of irradiation and on the dose of irradiation.

2001. Cultivation and titration of Japanese
B encephalitis virus in embryonated eggs.
Dorotuy G. SmitH AND ALBERT 8S. HERRING
(introduced by Grorce C. Wricut). Camp
Detrick, Frederick, Md.

Certain growth characteristics of Japanese B
encephalitis virus in embryonated eggs have been
investigated using embryo and mouse-brain prepa-
rations of six strains of the virus. The suscepti-
bility of 5- to 14-day eggs inoculated by either
the allantoic-cavity, yolk-sac or amniotic-cavity
routes was studied. Eggs were most susceptible to
infection by the yolk-sac route, regardless of the
age of the embryo, but the most uniform death
response occurred in the 10-day egg. When 10-
day eggs were inoculated into the yolk sac with
approximately 25,000 mouse IC LpD50/0.25 ml, the
majority of embryos died 48-96 hr. later. Some dif-
ferences were noted between strains in the death
pattern of infected embryos. At the peak of death,
the virus was 0.3 to 1.5 log higher in dead eggs than
in living ones. A method of titrating the virus was
developed using the yolk-sac inoculation of 10-day
embryonated eggs followed by 10 days incubation
at 35°C; this was compared with the recognized
mouse-intracerebral titration. The egg technique
was the more rapid assay and, on the basis of
volume inoculated, was also the more sensitive
measure of virus. Statistically, the yolk-sac
method of titration in eggs was approximately
as accurate as the mouse-intracerebral assay.

2002. Human plasmapheresis and its effect
on antibody levels. Jos—EpH SMOLENS AND
JosEPH STOKES, JR.* Children’s Hosp. of Phila-
delphia, and Dept. of Pediatrics, School of Medi-
cine, Univ. of Pennsylvania, Philadelphia.
Plasmaphresis is the process in which whole

blood, obtained from a donor, is continuously

separated into plasma and red blood cells; the
red blood cells being returned immediately to the
donor ang _the plasma retained. An ADL Cohn
blood fractionator is being utilized for biweekly
plasmaphereses of 23 donors. The phlebotomy
consists of 1 pt. of blood with about 200 ml of
plasma retained, the remaining 300-ml volume
being returned to the donor. Seventeen plasma-
phereses have been carried out on each of the
individuals thus far in 34 weeks. The following
tests are run at each plasmapheresis: red blood
cell, white blood cell, differential and platelet
counts, hemoglobin, hematocrit, sedimentation
rate and prothrombin time. Paper electrophoretic
pattern and nitrogen content are determined for
each plasma sample. Pertussis, mumps and
streptolysin O antibodies have been titrated in

FEDERATION PROCEEDINGS

Volume 16

each plasma sample since some of the donors have
been immunized versus either mumps of pertussis
antigens. A nonimmunized control group is also
included. The purpose of this antibody study is
to determine the effect of biweekly plasmapheresis
on viral, bacterial and nonviral nonbacterial anti-
bodies in man. All of the above blood tests have
remained within normal limits so far. Data will be
presented covering a full years’ study on the effect
of biweekly plasmapheresis on man and the effect
on antibodies.

2003. Blood group active substances of plant
origin. GrorGc F. SprinGER (introduced by
M. G. Sevaa). Pepper Lab., Univ. of Pennsyl-
vania, Philadelphia.

We found substances of high molecular weight
with blood group A, B and H(O) activity in higher
plants and bacteria grown in media contaning
salts-glucose only (Naturwissenschaften 42: 37,
1955). Animals were immunized with killed smooth
Escherichia coli Oss showing specificity for blood
group B only. Activity of this bacillus approx-
imates that of highly purified human B mucoids
against all sera tested. Similarly both Salmonella
poona and Taxus cuspidata twigs which were ex-
clusively H(O) active have been used. Blood group
specific immune antisera were obtained by either
parenteral or oral administration. A fraction from
Taxus cuspidata (fresh autumn twigs) precipitat-
ing at 55-60% ethanol concentration was con-
siderably more active (weight basis) than any
known mammalian H(O) mucoid in neutralizing
eel anti-H(O) serum, and somewhat less potent in
neutralizing anti-human H(O) rabbit immune
serum. This fraction is not inactivated by ‘Bifidus
enzyme’ (Federation Proc. 12: 901, 1953) and con-
tains less than 0.1% nitrogen, Paper chromatog-
raphic and colorimetric analyses of hydrolysates
revealed presence of rhamnose, arabinose, xylose
(little), galactose, glucose, fructose and one com-
ponent with Rr between that of tyvelose and
abequose (Angew. Chemie 65: 555, 1953). Another
fraction of somewhat lower serologic activity
showed an additional ‘fast component.’ Hexos-
amines and fucose were not demonstrable. Ultra-
centrifugal, electrophoretical and spectrophoto-
metrical data will be given. Relations of chemical
characteristics of these 3 materials to their speci-
ficity and the possible bearing of the existence of
plant blood group active substances upon the
problem of isoagglutinin formation will be dis-
cussed.

2004. Experimental typhoid fever in chim-
panzees. III. Pathogenesis. HELMUTH
Sprinz,* Maurice Lanpy, Smney Garnes*
AND GEOFFREY Epsa.u. Walter Reed Army Med.
Ctr., Washington, D. C.

The present study of a chimpanzee orally in-

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ipitat-
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lysates
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e com-
se and
nother
ctivity
Hexos-
Ultra-
photo-
emical
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nce of
yn the
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chim-
LMUTH
AINES*
y Med.

lly in-

March 1956

fected with Salmonella typh. Ty 2 and killed prior
to appearance of first clinical symptoms, is con-
cerned with the dissemination of bacilli and de-
velopment of specific pathology. Following oral
challenge, feces are cleared of bacteria within 24
hr., blood culture becomes positive 4-5 days post
challenge, and clinical symptoms develop shortly
thereafter. Consequently a chimpanzee was killed
96 hr. following oral challenge. At autopsy maxi-
mum numbers of bacilli were found in Peyer’s
patches, mesenteric lymph nodes, and thoracic
duct; smaller but significant numbers in the wall
of the ascending colon and spleen; a lesser number
in peripheral lymph nodes; few bacteria in blood,
liver and bone marrow; none in bile, feces, tonsil
and wall of small bowel outside Peyer’s patches.
Pathological examinations of tissues revealed
formation of specific typhoid granulomata in a
distribution similar to that seen in human in-
fections; however, intestinal ulcerations were not
observed. The findings indicate that initial multi-
plication of typhoid organisms takes place in
lymphatic tissues of the bowel, especially in
Peyer’s patches. Abdominal lymph nodes are
the other center of primary multiplication. The
thoracic duct is fed with infected chyle which is
discharged into the general circulation. The
generalized lymphadenopathy and splenic involve-
ment which occur are caused by hematogenous
spread of the infection. The findings in liver and
gall bladder indicate that the bile becomes in-
fected only secondarily after the bacteremia has
become well established.

2005. Mechanisms of antibody synthesis by
transplanted cells. ABRAM B. STAviTsky.
Dept. of Microbiology, Western Reserve Univ.
School of Medicine, Cleveland, Ohio.

It was reported previously that transplantation
of lymphoid cells from immunized to normal
animals resulted in the appearance of antibody
in the recipients. The present study was designed
to elucidate the mechanisms of antibody pro-
duction following homotransplantation. Rats or
rabbits were immunized with diphtheria toxoid or
bovine gamma globulin. Three days after the
second injection splenic or lymph nodal cells were
isolated and intraperitoneally transplanted. Anti-
body was measured by the Boyden hemagglutina-
tion or quantitative precipitin methods. Viable
transferred cells apparently were required for
antibody production since treatment with cy-
anide, sonic oscillation or x-irradiation abolished
antibody production. The process did not involve
transfer of enough antibody, gamma globulin or
total protein to account for the amount of anti-
body produced. In a typical experiment the cells
contained 4 ug gamma globulin and 45 yg total
protein, whereas the recipient contained 15,000

AMERICAN ASSOCIATION OF IMMUNOLOGISTS

615

ug of plasma antibody globulin at the peak of the
antibody response. Moreover, when cells were
labeled with S;; methionine and transferred, no
radioactivity appeared in the resulting antibody.
Nor was labeled normal gamma globulin incorpo-
rated into antibody in a typical transplantation
experiment. Further evidence for de novo syn-
thesis of antibody in the recipient was furnished
by injection of 83; methionine and its incorpora-
tion into antibody. Autonomy of the cells was
evidenced by production of antibody upon trans-
plantation of cells into recipients themselves
unable to make antibody by virtue of pantothenic
acid deficiency or immaturity.

2006. Electrophoretic and _ ultracentrifugal
studies of rabbit antiserum to sheep red
blood cells and to boiled sheep RBC stro-
mata. PETER STELOS* AND Davip W. TALMAGE.
Dept. of Microbiology and Medicine, Univ. of
Chicago, Chicago, Ill.

Ultracentrifugal analyses of rabbit antiserums
to sheep red blood cells (anti-RBC) and to heated
sheep RBC stromata (anti-F) indicate that the
major part of the hemolytic activity is found in
a globulin fraction having a sedimentation con-
stant of 18-20 Svedbergs. Starch electrophoresis
of anti-F serums indicates that hemolytic activity
is maximally concentrated between the 6- and
y-globulins. Thus, in terms of both centrifugal
and electrophoretic behavior, the major part of
Forssman hemolytic activity is contained in
globulins with similar properties to the T-com-
ponent found in certain horse antiserums. In
contrast, starch electrophoresis of anti-RBC
serums from which anti-F had been absorbed with
heated stromata (anti-isophile serum), showed
hemolytic activity to be associated mainly with
the y-globulin fraction. Anti-F serum globulins,
prepared by 50% ammonium sulfate precipitation,
were partitioned by starch electrophoresis. The
starch eluates were then tested for combining
capacity using the method of Talmage and Freter
(J. Infect. Dis. In press) and for hemolytic ac-
tivity. The maximum combining capacity was
found in the slower moving y-globulins while
hemolytic activity was centered, as described
above, between the 8 and y fractions. These find-
ings suggest that anti-F serum contains two types
of antibodies which differ in electrophoretic and
hemolytic properties.

2007. Neonatal serologic diagnosis of hemo-
lytic disease of the newborn (HDN) caused
by ABO incompatibility. Kurt Stern,
IsRAEL DaviIDSOHN AND ARNOLD BuzNITSKY.*
Dept. of Pathology, Chicago Med. School and
Mount Sinai Med. Research Fndn., Chicago, Til.
In analogy to the presence of Rh antibody in

cord blood of infants with HDN caused by ma-

616

ternal Rh sensitization, anti-A or anti-B anti-
bodies might be expected to appear in blood of
infants with HDN caused by ABO incompati-
bility. Occasional observations of such findings
have been reported by various authors, with a
more detailed search recently carried out by
Zuelzer and Kaplan (Am. J. Dis. Child. 88: 319,
1954). We have collected data on presence and
titer of complete and incomplete anti-A and anti-B
antibodies in cord bloods derived from a) homo-
specific pregnancies; b) heterospecific pregnancies
resulting in normal offspring; c) heterospecific
pregnancies resulting in offspring with HDN. In
11 of 13 infants of the last category, homologous
incomplete agglutinin (e.g., anti-A in A infants,
anti-B in B infants) was demonstrable, whereas
homo:ogous agglutinin was found in only 2 of 50
infanis of group b, of which one was a complete
antibody. For demonstration of the homologous
agglutinin in infants one or more of the following
technics was necessary: 1) indirect antiglobulin
test; 2) papain-treated erythrocytes; 3) combina-
tior. of 1 and 2. Results of these tests will be cor-
related with those of the direct antiglobulin test
on infant’s blood and of maternal isoagglutinin
studies, and with laboratory and clinical findings
in the infant. (Supported in part by a research
grant (A-77) from the Natl. Insts. of Health.)

2008. Antibody production in organ culture.
Kines.ey M. STEVENS AND JoHN M. McKEnna.*
Dept. of Virus Research, Sharp and Dohme, Div.
of Merck & Co., West Point, Pa.

Rabbits weighing 2.5 kg were given 20 mg of
alum-precipitated bovine serum albumin (AP
BSA) both intravenously and intraperitoneally,
followed 4-6 wk. later by 4 mg of AP BSA intra-
venously. Three hours later the spleens were re-
moved, cut into 2-mm cubes and 12 cubes planted
on tantalum gauze in each of several small cups
(modified from TRowELL, J. Exp. Cell Res. 6: 246,
1954). Sufficient Trowell’s Medium L at px 7.8
was added to keep the tantalum gauze moist and
incubation was carried out at 36.5° under 95%
02:5% COz. All cultures were set up in duplicate.
Antibody titrations on the fluid phases and on
saline extracts of the tissue were carried out using
Boyden’s hemagglutination technique (J. Exper.
Med. 93: 107, 1951). The antibody titers in the
fluid extracts remained constant and were never
>64. The titers in the fluid phases exceeded the
extract values after 6 hr. incubation, and reached
maximal values of 640 in 15-18 hr. When 3 days
elapsed between secondary injection and prepara-
tion of cultures, much higher antibody titers were
attained in the fluids after incubation. In all cases
antibody titers began to fall after 3 days in
culture.

2009. Immunogenetic studies of tryptophan
synthetase formation in Neurospora crassa.

FEDERATION PROCEEDINGS

Volume 16

Sriemunp R. Suskinp (introduced by A. M,

PAPPENHEIMER, JR.). Dept. of Microbiology,

New York Univ. College of Medicine, New York

City.

Rabbit antiserum has been obtained against
70-fold purified tryptophan synthetase, the en-
zyme which couples indole and serine to form
tryptophan in Neurospora. This antibody con-
pletely and quantitatively neutralizes enzyme
activity and the reaction is unaffected by the
presence of substrate. Several allelic tryptophan.
requiring mutants of Neurospora, unable to form
this enzyme, produce an immunologically related,
enzymatically inactive protein. This protein fol-
lows the same course of purification as the enzyme
and leads to the formation of anti-enzyme anti-
body in rabbits, which appears indistinguishable
from the antibody obtained against the enzyme
itself. The enzyme and the cross-reacting protein
in the mutants exhibit equal affinity for antibody,
However, this relationship is altered when the
two antigens are dialyzed. Enzyme, irreversibly
inactivated by dialysis, shows markedly reduced
affinity for antibody, active enzyme reacting pref-
erentially. On the other hand, the affinity of the
cross-reacting mutant protein for antibody remains
unaltered by dialysis. One mutant, lacking enzyme
and devoid of cross-reacting protein, has been
found. Immunization with extracts of such a mu-
tant elicits no anti-enzyme formation. Exhaustive
absorption of anti-enzyme antiserum with extracts
of this mutant removes other antibodies and gives
a serum specifically directed against the enzyme
or the cross-reacting mutant protein. Quantita-
tive neutralization and precipitation experiments
suggest that such serum may contain only a single
antibody.

2010. Direct determination of nuclease ac:
tivity in guinea pig epidermis. JOSEPH
TABACHNICK AND Emity CERcEO (introduced
by CuHarues Wess). Dept. of Microbiology,
Albert Einstein Med. Ctr., Northern Div., Phila
delphia, Pa.

The present procedures for determining nuclease
activity in tissues utilize indirect methods in
which yeast RNA or calf thymus DNA is usually
used as substrates. Using the u.v. method pre
viously described (Federation Proc. 14: 479, 1956)
the rate of hydrolysis of native RNA and DNA
was followed in guinea pig epidermal homogenates,
as an index of nuclease activity. At 37° the pi
optimum for RNase and DNase was about 7.2
To avoid losses of from 15-30% of the initial RNA
during homogenization, the homogenates (l¢
20%) were made in dilute acidulated saline. The
reaction was started by the addition of 0.1 ml of
0.6 m buffer. About 60% of the native epidermal
RNA was depolymerized in 10 min. at 37°. DNA
was hydrolyzed more slowly,—about 40% in 1 br.

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A. M,
abiology,
ew York

against
the en-
to form
ly com-
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ne anti-
uishable
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hen the
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JOSEPH
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nuclease
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; usually
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ial RNA
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March 1956

The rapid. RNA loss was shown to be due to en-
zymatic degradation, being partially inhibited
by heat as well as by known RNase inhibitors,
such as heparin and sodium polyanhydroman-
nuronic acid sulfate (Paritol M, Wyeth). In addi-
tion there was a rapid rise in acid soluble organic
P which corresponded to the loss of RNA, while
the inorganic P showed little increase (at px 7.2)
until about 80% of the RNA had been degraded.
Under our experimental conditions the epidermal
RNA and its hydrolase(s) appear(s) to be on the
same particle. This was suggested by a simple
dilution experiment which resulted in no lowering
in RNase activity when a 10X diluted homogenate
was compared with the undiluted control. Pre-
liminary experiments showed the high RNase
activity of epidermis to be unique for this con-
tinually proliferating tissue. Other guinea pig
organs such as liver, kidney and spleen gave rates
for native RNA hydrolysis which were about 35%
lower than those for epidermis. (Supported by a
grant from the Atomic Energy Commission con-
tract AT (30-1)-1727.)

2011. Some viral susceptibilities of human
amnion cells in tissue culture. KENNETH K.
TAKEMOTO AND ALBERT M. LERNER (introduced
by Doruanp J. Davis). Lab. of Infectious Dis-
eases, Natl. Microbiological Inst., NIH, Be-
thesda, Md.

The multiplication of Types I, II, and III polio
virus in human amnion cells has been reported by
Fogh et al. (Science 122: 30, 1955). Since human
amniotic membranes provide a cheap, readily
available source of cells for tissue culture, the
susceptibility of these cells to a number of other
viruses was studied. Collection and trypsinization
of membranes was essentially similar to that de-
scribed by Fogh et al. The yield of cells per mem-
brane after trypsinization varied from none to
1.3 ml with an average of 0.5 ml. The reason for
this variation has not been determined. In Eagle’s
medium consisting of amino acids, vitamins,
glutamine, salts and 10% horse serum, good
growth was obtained by 5-7 days and cultures
could be maintained for several weeks on this
medium. Cytopathogenic effects and viral multi-
plication were observed with the following viruses
which were carried through 5 successive passages:
Adenoidal-pharyngeal-conjunctival (APC) viruses
types 1 through 8, Coxsackie virus (1 type A and
2 type B strains), and herpes. Viruses which
showed no evidence of growth either by cyto-
pathogenicity or tests of passage fluids in other
susceptible hosts were the hemagglutinating
viruses influenza A, B, C, mumps, and newcastle
disease virus, and also dengue.

2012. Ultracentrifugal separation of two
antibodies of similar specificity but dif-
ferent hemolytic efficiency. Davip W. Tat-

DS ae wa ae

AMERICAN ASSOCIATION OF IMMUNOLOGISTS

617

MAGE, GLorIA L. FRETER* AND WILLIAM H.
TALIAFERRO. Depts. of Medicine and Micro-
biology, Univ. of Chicago, Chicago, Iil.

The number of 50% hemolytic units in antisheep
red cell serums has been compared with their con-
tent of antibody as measured by a competitive
absorption test with I'*! trace-labeled antibody.
This absorption test was developed to measure
the capacity of an antiserum to combine with a
red cell antigen. One combining unit has been
arbitrarily defined as that amount of serum which
produces 50% inhibition in the uptake of labeled
antibody by a standard number of red cells. In
addition to sensitivity and simplicity, the com-
petitive absorption test has the advantage that
the specificity of the antibody measured may be
limited to the specificity of the labeled antibody.
Six serums from rabbits immunized with heat red
cell stromata (anti-Forssman) and two anti-red
cell serums absorbed with heated stromata (anti-
isophile) were centrifuged for 200 min. at 105,00 x
g. In all cases, the hemolytic efficiency (ratio of
hemolytic to combining units) of the fractions
obtained increased progressively from top to
bottom. The average increase in hemolytic ef-
ficiency was 10-fold. These findings suggest the
presence of at least 2 antibodies with different
physical and hemolytic properties in both the
Forssman and isophile antiserums. The hemolytic
efficiency of the antibody is thought to be de-
pendent on some physical property of the anti-
body molecule (probably size) rather than some
minor difference in specificity because of a) the
magnitude of the difference in hemolytic efficiency;
b) the similarity of the centrifugal separation with
Forssman and isophile antiserums; and c) the
similarity of the decline in the hemolytic and
combining units when Forssman antiserums were
absorbed with varying numbers of sheep red cells.

2013. Effect of ultraviolet light on polio-
myelitis virus. ALton R. Taytor, W. W.
Kay,* E. A. Trum,* I. W. McLzan, Jr., F.
OPppPENHEIMER* AND F. D. Strmpert.* Research
Labs., Parke, Davis & Co., Detroit, Mich., and
Michael Reese Research Fndn., Chicago, Ill.

The effect of ultraviolet irradiation (2537A)
upon all 3 strains of poliomyelitis virus (Mahoney,
Type I; MEF-1, Type 2; and Saukett, Type 3) in
Medium 199, and upon purified virus suspensions
in saline solutions has been studied. The rate of
inactivation of the virus was determined using
6-watt increments of ultraviolet incident energy
in an Oppenheimer ‘Centri-Filmer.’ None of the
curves obtained was exponential in either Medium

_ 199 or in saline suspensions. Factors other than

the primary photochemical inactivation reaction
are known to be operative and to affect the course
of the inactivation of the virus as measured by
biological titration. Medium 199 exhibits marked

618

ultraviolet absorption in the region of 250-270
my. Following progressive irradiation, the charac-
teristic absorption spectrum of the virus (maxi-
mum absorption at 260 mz, minimum at 238 mz)
undergoes a drastic change. The maximum peak
shifts from 260 my to 250 my and there is an in-
crease in overall absorption, especially in the
region of 225-250 mu. Virus showing such altered
spectra is capable of producing good antibody
response, but incapable of multiplication in tissue
culture or animals. The same type of relationship
was studied for equine encephalomyelitis virus
(Tayuor et al. J. Infect. Dis. 69: 224, 1941) and
a decrease in inactivation rate with progressive
irradiation shown to be due to an increase in the
ultraviolet absorption of the virus. This demon-
strative change in the absorption of poliomyelitis
virus can be correlated with consistent negative
safety tests in tissue culture.

2014. Serological approach to epidemiology
of typhus and Q Fever in Bosnia. A. L.
TeRzIN AND J. Gaon (introduced by F. S.
CHEEVER). Med. Faculty, Univ. of Sarajevo,
Sarajevo, Bosnia.

One hundred and fifteen serum samples from
healthy Bosnians belonging to three different age
groups (pre-school children, adolescents and
adults over 50 yr.) were tested for complement
fixing antibodies to typhus and Q fever antigens.
The serological results, when correlated with
certain living habits of the local Moslem and non-
Moslem population groups, permit the following
conclusions: a) Although infested with body lice
at approximately the same rate as the non-
Moslems, the Moslems appeared to be more ex-
posed to typhus infection in childhood than are
the non-Moslems, and thus the incidence of typhus
in childhood is greater in Moslems. b) The rate of
typhus infection in Moslem children is approx-
imately the same as that in Moslem adults. c) By
contrast, non-Moslem children are infected with
typhus at a lower rate than non-Moslem adults.
d) While typhus infection in the Moslem popula-
tion below the age of 20 yr., and especially in
children, is primarily in the epidemic form, typhus
infection in non-Moslems occurs in all age groups,
in sporadic as well as epidemic forms. e) Infection
with Q fever takes place in the non-Moslem pop-
ulation at an earlier age and with a higher infec-
tion rate than in the Moslems. The incidence of
Q fever positive sera in Moslem children is some-
what lower and the rate of increase of positive
sera by age is slower than in non-Moslems. These
findings are explained on the basis of the different
customs and habits in these two groups of the
population.

2015. Necrotizing action of adrenaline in
endotoxin-treated animals. LEw1s THOMAS.

FEDERATION PROCEEDINGS

Volume 16

New York Univ. Dept. of Pathology, Bellevue

Med. Ctr., New York City.

Certain physiological alterations produced in
rabbits by the shock-producing, pyrogenic endo-
toxins (somatic antigens) of gram negative bac-
teria resemble systemic effects of adrenaline,
These include intense peripheral vasoconstriction
(followed terminally by vasodilatation), hyper-
glycemia followed by hypoglycemia, depletion
of liver glycogen, and elevated blood lactate and
pyruvate levels. To learn whether endotoxin
affects the reactivity of peripheral vessels to
adrenaline, rabbits were injected intravenously
with small amounts of highly purified EZ. coli or
E. typhosa endotoxin. Simultaneously, various
doses of adrenaline (in 0.2 cc) were given intra-
cutaneously in the abdominal skin. Within 12-18
hr., extensive flat lesions of hemorrhagic necrosis,
5-8 cm in diameter, developed at the injected sites,
The minimal effective doses of endotoxin were
5 wg, of adrenaline 10 ug. Mixtures of endotoxin
and adrenaline were injected intradermally with
similar results, except that hemorrhagic necrosis
involved a smaller area and was accompanied by
marked local inflammation. These lesions re-
sembled typical Shwartzman reactions. The re-
actions were reproduced by nor-adrenaline, but
not by ephedrine, Pitressin or serotonin. They
were inhibited by chlorpromazine, but not by
heparin, cortisone or nitrogen mustard. Nitrogen
mustard greatly enhanced the severity of necrosis
caused by mixtures of endotoxin and adrenaline,
suggesting an inhibitory affect of leucocytes on
the necrotizing action of adrenaline. Similar
phenomena were observed in mice, rats and
hamsters. In mice, sublethal doses of adrenaline
caused convulsions and death when given 30 min.
after endotoxin. In summary, endotoxin causes
intense vascular hyperreactivity to adrenaline.
This phenomenon may be of importance in the
pathogenesis of endotoxin shock, and in the
Shwartzman reaction.

2016. Dynamies of formalin inactivation of
poliomyelitis virus suspensions. EvGENE A.
Timm,* I. W. McLean, Jr., C. H. Kupsky* anb
A. E. Hoox.* Research Labs., Parke, Davis &
Co., Detroit, Mich.

Formalin inactivation curves were determined
for tissue culture suspensions of Mahoney, MEF-1,
and Saukett strains of poliomyelitis virus by
plotting tissue culture titers of samples of the
virus suspension being treated against total time
of treatment. Against final formalin concentra-
tions of 1:2000, 1:4000, and 1:8000 (dilution of
37% formaldehyde solution) in filtered suspen-
sions, the MEF-1 strain was the most stable and
the Saukett strain the least stable. The inactiva-
tion curves showed that the drop in titer during

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the initial ‘stage of inactivation was dispropor-
tionately high, the last 10% or less of active virus
requiring 30 to over 100 times as much time for
complete inactivation as the first 90% or more.
The resulting curves, therefore, did not conform
to a first order reaction. Curves determined for
inactivation at 20°C and 40°C and for inactiva-
tion at pu 6.5, 7.0, 7.5 and 8.0 showed that this
initial high drop in titer varies only in degree with
change in temperature and pu. The initial titer
loss is less marked at 30°C than at the higher tem-
peratures. Similarly, when the pH was nearer
neutrality this drop was less marked. All of the
above results were based on the addition of the
formalin to pre-warmed suspensions, the time of
this addition being considered as zero time. Con-
trol tests showed that when formalin was added
to cold suspensions and zero time was taken as
the time at which the formalin-virus suspension
reached the test temperature (usual in production
practice), the initial disproportionately high drop
following zero time was still readily detectable.
Variable results observed routinely in large scale
formalin inactivation can be attributed at least
partially to variation in the inactivation rate and
the non-exponential nature of the inactivation by
formalin.

2017. Immunological aspects of bacterio-
phage-host cell interaction. L. J. TouMacH
(introduced by JoHNn R. Cann). Dept. of Bio-
physics, Univ. of Colorado Med. Ctr., Denver.

It has been known that neutralization of bac-
teriophage by specific antiserum affects a step
occurring early in the phage reproductive cycle.
Recent experiments of Nagano and Mutai (Compt.
rend. soc. biol. 148: 757, 1954) which showed that
attachment of T2 phage to its host cell is scarcely
inhibited by neutralization, and of Nagano et al.
(Japan. J. Exptl. Med. 22: 145, 1952) which showed
that neutralized phage does not kill the cell to
which it attaches, have further localized the in-
hibition. The foregoing results have been in-
dependently confirmed with T2, and extended to
Tl. Further, it has been shown that neutralized
T2 fails to eject its DNA after attaching, and that
once attached, can easily be eluted. Therefore
irreversible attachment is blocked. Other studies
with active phage indicate that the irreversible
bond is extremely stable, and that its formation
is a prerequisite for subsequent steps of the pene-
tration sequence. The phage tail, which is its at-
tachment organ, bears the antigenic sites at which
neutralization occurs (LANNI AND LANNI. Cold
Spring Harbor Symp. Quant. Biol. 18: 159, 1953),
but the capacity of phage to combine with neu-
tralizing antibody is not eliminated after ir-
reversible attachment to host cells. This suggests
the existence of similar antigenic structures on a
large portion of the tail sheath.

AMERICAN ASSOCIATION OF IMMUNOLOGISTS

619

2018. Production of delayed sensitivity to
protein without detectable circulating
antibody. J. W. Uur,* 8. B. SALvIN AND A. M.
PAPPENHEIMER, JR. Dept. of Microbiology, New
York Univ. College of Medicine, New York City.
Specific precipitates were prepared by addition

of purified diphtheria toxoid to an excess of anti-

toxin (rabbit, human, horse and guinea pig).

Washed precipitates (of composition TA,;) con-

taining 2.5 wg of toxoid were incorporated in

Freund’s adjuvant and injected into guinea pigs.

Regardless of the route of injection, the animals

when skin tested 7-16 days later with 0.3 wg or

less of purified toxin and toxoid showed marked
delayed hypersensitivity of the tuberculin type.

The sensitized animals were Schick positive and

their serum contained less than 0.001 vu antitoxin/

ml. No symptoms of anaphylactic shock could be

elicited even after intravenous injection of 2 mg

toxoid. Sensitivity could be transferred to normal
guinea pigs with lymphocytes from the regional
nodes of sensitized animals. Mycobacteria are not
necessary to produce sensitization by this method.

Specific precipitates suspended in Arlacel and

Bayol F mixtures, however, produced a higher

degree of sensitivity than saline suspensions of the

specific precipitates. Pronounced sensitivity was
induced by intraperitoneal injection of iarge

amounts of antitoxin followed 24 hr. later by 3

yg toxoid in oil into the footpads. Control animals

injected with 2.5 ug purified toxoid in oil showed

Arthus reactions when skin tested 16 days later;

they were Schick negative and readily developed

anaphylactic shock after intravenous injection of
toxoid. The method appears to be a general one,
since delayed sensitization to crystalline egg
albumen may be produced by injection of similarly
prepared specific precipitates in antibody excess.

2019. Zine, a component of alcohol dehy-
drogenase of horse liver. Bert L. VALLEE
AND FrReEpERIC L. Hocu (introduced by GEORGE
P. Berry). Biophysics Research Lab., Dept. of
Medicine, Harvard Med. School and Peter Bent
Brigham Hosp., Boston, Mass.

Emission spectrographic and microchemical
analysis of highly purified yeast ADH has shown
it to contain 4 moles of zinc. The molecular weight
of yeast ADH is 150,000; it binds 4 moles of DPN.
The molecular weight of horse liver ADH is 73,000
and it binds 2 moles of DPN. These findings
prompted us to suggest (VALLEE, B. L. anp F. L.
Hocu, Proc. Natl. Acad. Sci. 41: 327, 1955) that the
horse liver enzyme contains 2 moles of zinc.
Nygaard and Theorell, (Acta chem. Scand. 9: 1241,
1955), have verified that the purified enzyme con-
tains zinc. Thus far, no detailed studies on the
metalloenzyme characteristics of the liver ADH
have been reported. Spectrographic, microchemi-
cal, polarographic and enzymatic examination of

620

horse liver ADH demonstrate a concomitant rise
of zinc and enzymatic activity with increasing
purification. The zinc content approaches 2 moles
of zinc/mole of enzyme in highly purified crystal-
line horse liver ADH. Simultaneously, extraneous
metals are removed. The enzyme is inhibited by
chelating agents such as orthophenanthroline,
8-hydrooxyquinoline, and sodium diethyldithio-
carbamate. The inhibition is a function of temper-
ature, time, pH and concentration of inhibitor.
The molecular configuration and dentate structure
of the chelating agents employed condition the
inhibition significantly. The effects of DPN and
DPNH on the inhibition by chelating agents will
be discussed. Zine is firmly bound to the apo-
enzyme, and the active complex has been assigned
the empirical formula [(ADH)Zn2] (DPN):.

2020. Structure of T2,;+ bacteriophage pro-
tein. HELEN VAN VUNAKIS AND James L.
BaRLow (introduced by Jessre L. HENpRy).
Div. of Labs. and Research, New York State Dept.
of Health, Albany.

Bacteriophage T:** was grown in glucose syn-
thetic medium in 100-liter lots. It had a final titer
of 4-7 X 10" infective U/ml. (BARLOW AND VAN
Vounakis. Annual Report, Div. of Labs. and Re-
search, 1955). The purified virus was split into its
protein and nucleic acid moieties by ‘osmotic
shock.’ The protein, freed of nucleic acid, was
isolated by precipitation at low ionic strength
at pH 4.4 followed by differential centrifugation.
Good quantities of purified virus protein were
thus made available for structural studies. Using
the dinitrofluorobenzene technic of Sanger, the
N-terminal amino acid of the protein was de-
termined and found to be alanine. If the virus
protein is considered to be homogeneous and the
isolated DNP alanine is only a single residue, then
alanine would occupy the N-terminal position in
a protein chain of approximately 80,000 M.W.
Complementary experiments to determine the
C-terminal residue(s) using carboxypeptidase di-
gestion are being carried out at present. Carb-
oxypeptidase did not have any effect on the infec-
tivity of thé intact virus or on the lytic properties
of the viral protein. The significance of these re-
sults in view of what is already known from physi-
cochemical and immunologic studies about the
structure of the protein coat will be discussed.

2021. Behavior of agglutination activating
factor of rheumatoid arthritis sera with
immune precipitates. JoHN H. VauGHAN.
Med. College of Virginia, Richmond.

The agglutination activating factor (AAF) in
the sera of individuals with rheumatoid arthritis
is capable of agglutinating several types of cells
sensitized with antibody of various animal species.
This has suggested that its behavior may be more

FEDERATION PROCEEDINGS

Volume 16

like that of serum complement than that of
serum antibody and therefore that it might be
profitably studied with immune precipitates.
Washed precipitates from sera of rabbits im-
munized with recrystallized egg albumin (Ea)
or conalbumin (Ca) were added to heat-inacti-
vated rheumatoid and normal human sera. In-
creasing quantities of precipitate (20-250 ug N)
absorbed increasing quantities of N from rheuma-
toid arthritis sera (9-60 ug/ml serum GN), a
plateau being reached at and beyond 25 ug of
precipitate N. Successive absorptions with 250
ug of precipitate N, however, provided some ad-
ditional absorbable N; total N absorbable from
serum GN was 99 ug. No N was absorbed from in-
dividual or pooled normal sera. With the absorp-
tion of N from the rheumatoid arthritis sera, the
AAF activity of the supernatant was first reduced
and then abolished completely. In control studies,
powdered asbestos, filter paper, and barium
sulfate did not absorb any measurable N nor re-
duce the AAF titer. Guinea pig and horse Ea-anti
Ea and diphtheria toxin-human antitoxin also
failed to absorb AAF. Heating rheumatoid ar-
thritis sera for 4 hr. at 56°C reduced the AAF titer
and absorbable N only slightly. Simultaneous
analyses of precipitates by Kjeldahl, Biuret, and
Folin-Coicalteu reactions give corresponding
values. The anthrone reaction indicates the ad-
dition of a carbohydrate moiety.

2022. Biological conversion of cholestenone-
4-C“% to an unsaturated 38-OH sterol.
Heven §S. VisHnrac AND Faitu J. NIELSEN
(introduced by H. P. Trerrers). Microbiology
Dept., Yale Univ., New Haven, Conn., and
Biology Dept., Brookhaven Natl. Lab., Upton,
MN, 7;

Sterol containing C™ was isolated from Labyrin-
thula vitellina var. pacifica grown with choleste-
none-4-C" as growth factor. The specific activity
(cpm/mg C) of crystalline sterol (combusted and
counted as BaCO;) prepared after each purifica-
tion procedure in two experiments was prepara-
tion and regeneration of the digitonide—176 and
368; preparation and regeneration of the bromide—
173 and 377; chromatography on alumina and
elution with petroleum ether:ethyl ether—1i7
and 350 respectively. Although only about 1% of
the total activity supplied as cholestenone was
recovered as sterol (of markedly lower specific
activity), the absence of significant changes in
specific activity on purification of the sterol indi-
cates that the sterol itself contained C™, The
time course of the Liebermann-Burchard reaction
identifies this sterol as a 38-OH sterol unsaturated
at C5, probably cholesterol. The advice and as-
sistance of Drs. M. Gibbs and W. M. Stokes are
gratefully acknowledged. (Aided by a contract

eS Ora $3 =

=>
=

ne 1§

at of
it be
rates,
> im-
(Ea)
1acti-
. In.
ig N)
uma-
N), 8
ug of
h 250
e ad-
from
ym. in-
)SOrp-
a, the
duced
udies,
arium
or re-
a-anti
1 also
id ar-
* titer
neous
t, and
nding
1e ad-

none.
terol,
ELSEN
yiology
, and
TJ pton,

byrin-
oleste-
tivity
>d and
rifica-
epara-
76 and
nide—
a and
r—157
1% of
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pecific
ges in
1 indi-
4 The
action
urated
nd as-
ces are
mtract

March 1956

between Yale Univ. and the Office of Naval Re-
search, Dept. of the Navy, NR 135-241.)

2023. Immunologic study of rabbits with
diphtheritic polyneuritis. Byron H. Waks-
MAN, Raymonp D. Apams* anp Herpert C.
MaANSMANN, JR.* Depts. of Bacteriology and
Neurology, Harvard Med. School, and Massa-
chusetis Gen. Hosp., Boston.

Diphtheritic polyneuritis, produced in rabbits
by intravenous injection of subneutralized toxin-
antitoxin mixtures, is a neurologic disease ap-
pearing 2-4 wk. after the injection and progressive
over a period of weeks. Histologically it shows
segmental myelin destruction in spinal ganglia,
nerve roots, and to a limited extent peripheral
nerve, closely resembling the lesion of human
disease. No focal or inflammatory lesions are
present comparable to those of experimental
allergic neuritis or infectious disease of periph-
eral nerve. In the same animals one finds vig-
orous production of circulating antitoxin, ap-
pearing at about 2 wk. and maximal at 3-4 wk.,
and infrequently complement fixing antibody to
horse serum proteins. No antibody against rabbit
spinal cord or sciatic nerve was demonstrated.
Skin reactivity of the delayed type to both rabbit
nerve suspension and diphtheria toxoid appeared
during the 2nd week and was maximal in the 3rd,
ie., well before the peak of antitoxin response.
The circulating complement level showed a slight
increase at 2-3 wk. No correlation existed between
the development of diphtheritic polyneuritis and
any of these immunologic events, either in time or
in degree. Treatment of rabbits with 400 r whole
body irradiation 48 hr. before injection resulted
in severe leukopenia lasting about 3 wk., and
delay of antitoxin formation with considerable
reduction of peak titers. It has little effect on the
development of skin reactivity and apparently
none on the disease process. It is concluded that
diphtheritic polyneuritis is produced by a non-
immunologic mechanism, e.g. direct toxicity.

024. Growth and titration of vaccinia virus
in embryonated eggs. Mary Surpp Watson,
HELEN JEFFRIES, JAMES W. Pryor AND Dor-
otuy G. SmitH (introduced by Grorce C.
Wricut). Camp Detrick, Frederick, Md.

An investigation has been made of methods for
wltivating and titrating three strains of vaccinia
virus in embryonated eggs. The susceptibility of
5- to 14-day eggs inoculated by one or four routes
was determined, namely, the yolk-sac, allantoic
tavity, amniotic cavity, or chorio-allantoic mem-
brane (CAM) route. Eggs were highly susceptible
to infection by all routes tested, regardless of the
age of the embryo. Death occurred most con-
tistently in 8-day eggs inoculated by the yolk-sac
toute and in 12-day eggs, by the CAM. When eggs

AMERICAN ASSOCIATION OF IMMUNOLOGISTS

621

were inoculated in this manner with about 1000
LDso, the majority of embryos died 3-4 days later
with no significant differences between the strains
of virus. High susceptibility to infection without
death was also indicated by pock formation on the
CAM following inoculation of 12-day eggs onto
the CAM. A comparison was made of the above
three procedures for assay of the virus. The most
practical and consistent method of titration was
a measure Of LDso using the 8-day egg inoculated
by the yolk-sac route followed by incubation at
35°C until death or for 10 days. Statistically, this
method was at least as sensitive and accurate as
the pock count and more accurate than that meas-
uring LDso following CAM inoculation.

2025. Role of magnesium in inhibition of
Newcastle Disease virus by the properdin
system. RautpH J. Wepawoop,* Harotp S.
GINSBERG AND Louis PitLEMER. Western Re-
serve Univ. School of Medicine, Cleveland, Ohio.
Inhibition of Newcastle Disease virus (NDV)

by fresh normal human serum requires properdin,
all four components of complement and mag-
nesium. These components of serum constitute the
properdin system which has also been shown to
have bactericidal and hemolytic activities. All
of these functions of the properdin system require
temperatures above 20°C for optimal activity.
Inhibition of NDV by the properdin system can
be limited by magnesium concentration. The re-
lationship between inhibition and magnesium
concentration appears to be a first order reaction.
Viral activity can be released from the inactive
virus-serum mixture by the removal of mag-
nesium. The reappearance of viral activity was
temperature dependent. The implications of these
findings as they apply to the nature of the inter-
action between the properdin system and virus
will be discussed.

2026. Release of I! complement from im-
mune precipitates. Witt1amM O. WEIGLE*
AND Paut H. Maurer. Dept. of Pathology,
Univ. of Pittsburgh School of Medicine, Pitts-
burgh, Pa.

Attempts to ‘saturate’ specific precipitates with
various complements as measured both by the
nitrogen taken up by the precipitates and the
disappearance of hemolytic activity from the
added complement led to ‘dissociation’ studies
with I!3! complement (I!*!C). I!81C which was pre-
pared by labeling the euglobulin fraction of guinea
pig serum was added to bovine serum albumin—
rabbit anti bovine serum albumin mixtures. When
fresh complement was added to the washed im-
mune precipitates, which contained I!*!C, 50-60%
of the I'*!C was released into the sera. Approx-
imately 30-40% of this released (‘dissociated’)
T31C could again fix to fresh immune precipitates.

622

Sera decomplemented with specific precipitates
or by heat (56°C), rabbit gamma globulin and
rabbit serum albumin solutions all caused a ‘dis-
sociation’ (15-37%) of I'**C. However, only 10-20%
of the labeled substance so ‘dissociated’ could
again fix to immune precipitates. NaCl (0.15 m)
released only 2-6% of the I'*C from immune pre-
cipitates. After antibody-antigen-I''C precipi-
tates were incubated with decomplemented serum
or with purified protein solutions subsequent in-
cubation with fresh guinea pig serum could still
‘dissociate’ 50-60% of the I!*C. However, 65%
of this ‘dissociated’ I'*!C could then fix to fresh
precipitates. These and related experiments will
be discussed. (Supported by A.E.C. Contract
No. AT (30-1)—1205.)

2027. Serum’ glycoprotein concentrations
during the anamnestic response. HENRy E.
WEIMER AND Eric L. NELSON (introduced by
A. F. Rasmussen, Jr.). Dept. of Infectious Dis-
eases, Univ. of California Med. Ctr., Los Angeles.
A previous study (J. Immuno.. 74: 248, 1955)

has demonstrated that following primary im-

munization in the guinea pig, statistically sig-

nificant increases occur in the levels of total serum
glycoprotein, seromucoid, and y-globulin poly-
saccharide. The present communication reports
similar observations made during the anamnestic
response. Twenty-eight adult, male guinea pigs
were immunized against an antigen prepared from
heat-killed Brucella suis cells. Following primary
immunization the agglutinin titer was allowed to
decline for 2 months; .5 ml of a 1.0% suspension
of the bacteria was then administered intraperi-
toneally. The animals were bled 3 days prior to
antigen administration and 4, 8, 12, 18, 24 and 46
days following. Agglutinin titers, total glyco-
protein, seromucoid, y-globulin polysaccharide,
total protein and y-globulin protein were de-
termined in serum specimens. Hemoglobin and
hematocrit levels were measured in oxalated blood
samples. To evaluate the effect of the repeated
bleedings, 18 normal guinea pigs were bled at
weekly intervals over a period of 5 wk. and the
same determinations were made. The maximum
immune response occurred 12 days following anti-
gen administration. Significant increases were
observed for the total serum glycoprotein and
seromucoid values 4 and 8 days respectively after
antigen administration. At the height of the anti-
body response the values had declined to the
normal range; y-globulin levels were also sig-
nificantly elevated. The increase in the poly-
saccharide moiety was greatest 4 days before the
agglutinin titers attained their highest values.

Repeated bleedings would not account for the

changes observed in the immunized group.

2028. Effect of tuberculin on tissue cultures
of the corneas of tuberculin-sensitive

FEDERATION PROCEEDINGS

Volume 1§

guinea pigs. RusseLL 8. WEISER AND Karz J,
May.* Dept. of Microbiology, Univ. of Washing-
ton School of Medicine, Seattle.

Roller tube tissue cultures were prepared from
the corneas and spleens of albino guinea pigs made
highly sensitive to the intracutaneous and intra-
corneal injection of tuberculin by infection with
the BCG strain of M. tuberculosis. Good growth of
corneal epithelium was obtained in a medium con-
taining 40% horse serum, 20% chick embryo ex-
tract, and 40% Hanks’ balanced salt solution con-
taining penicillin and streptomycin. Whereas the
incorporation into the medium of OT 1:20 or PPD
in a concentration of 0.02, 0.1 and 0.2 mg/ml
markedly inhibited the migration end growth of
the cells of splenic explants of sensitive animals,
the growth of corneal epithelium from sensitive
animals was not inhibited.

2029. Quantitative studies in hemagglutina-
tion. III. Further characterization of reac-
tion of antigens and antibodies of human
isohemagglutinins. M. H. WILKIE (intro-
duced by E. L. Becker). Walter Reed Army
Inst. of Research, Walter Reed Army Med. Ctr.,
Washington, D. C.

By means of quantitative hemagglutination
technique reported earlier, the human red cell-
isoagglutinin complex has been further investi-
gated. When fresh human serum containing anti-B
isoagglutinin was heated, treated with versene,
or absorbed with antigen-antibody precipitate,
there was the same marked fall in the agglutinat-
ing power of the serum whether measured at 4°,
25°, or 37°C. From this it is concluded that, under
our conditions of assay, complement has an en-
hancing action upon isohemagglutination. To
what degree the difference between this conclusion
as to the effect of complement and that of the
Wurmser group can be explained by a difference
in assay procedures, is under investigation. Con-
trary to our previous findings with A; sera, anti-B
agglutinin in group O sera apparently failed to
reach a true equilibrium when equilibrium was ap-
proached from two directions with a centrifuge
technique and a constant shaking method. Re-
moval of complement or changing the temperature
failed to affect this apparent lack of equilibrium.
However, absorption of anti-A from this serum
by group A, cells had a slight effect on equilibrium
and markedly diminished the anti-B component.
Supernatant studies of the two different equilib-
rium levels showed significant differences in the
amount of antibody remaining free at equilibrium.
In the centrifuge technique which produced mole
agglutination, less antibody was fixed by the cells
than the shaking technique with less agglutina-
tion.

2030. Inhibition of growth of pleuropneu-
monia-like organisms by anti-corynebac-

etc
(c)
syl
ent
diy
foo
inj
No
me
me
me
ale
du
ser
duc
pre

cul
Th
acii
for’
glu

ime 1§

ARL J,
shing-

| from
made
intra-
1 with
wth of
n con-
yO ex-
n con-
as the
r PPD
ng/ml
vth of
imals,
sitive

jtina-
reac-
uman
intro-
Army
. Ctr,

ration
1 cell-
vesti-
anti-B
rsene,
itate,
tinat-
at 4°,
under
in en-
1. To
lusion
of the
2rence
, Con-
anti-B
led to
‘as ap-
rifuge
1. Re-
rature
rium.
serum
brium
onent.
juilib-
in the
yrium.
| mole
e cells
utina-

pneu:
ebac-

March 1956

terium serums. Ruts G. WITTLER (introduced

by Joun F. Kent). Walter Reed Army Inst. of

Research and Veterans Admin., Washington, D.C.

The growth of various pleuropneumonia-like
organisms (PPLO) was shown to be inhibited
specifically by the incorporation of inactivated
homologous antiserum in the culture medium
(EDWARD AND FirzGERaLp. J. Path. Bact. 68: 23,
1954). During experiments employing this pro-
cedure, it was found that antiserum prepared to
human genital strains of Corynebacterium was
capable of inhibiting the growth of numerous
human genital strains of PPLO. These PPLO
strains were subsequently shown to be capable
of transformation to Corynebacterium species
(WiTTLER et al., in preparation). It has since been
found that antiserums to the human Coryne-
bacterium strains are also capable, although to a
lesser extent, of inhibiting the growth of various
animal strains of PPLO. The latter PPLO are
strains that have been in stock culture in various
laboratories for several years and have never been
suspected of bearing a genetic relationship to
any known bacterial genus. This finding suggests
that the growth inhibition test may be a significant
tool for the elucidation of the generic origins of
‘fixed’ PPLO strains which appear to be incapable
of transformation to recognized bacterial genera.

2031. Mechanisms of in vitro synthesis of
antibody by tissues of immunized animals.
BENJAMIN Wo tF* AND ABRAM B. STAVITSKY.
Dept. of Microbiology, Western Reserve Univ.
School of Medicine, Cleveland, Ohio.

It was observed previously (J. Immunol., 75:
214, 1955) that lymph node and spleen fragments
from immunized rabbits produce diphtheria anti-
toxin in vitro. The effects of physical agents (sonic,
etc.) and of inhibitors of oxidative metabolism
(cyanide, dinitrophenol) indicate that antibody
synthesis requires viable cells and a source of
energy. In the present study, alum-precipitated
diphtheria toxoid was twice injected into the rear
foot pads of rabbits. Three days after the booster
injection the regional lymph nodes were removed.
Node preparations were placed in Fisher’s (V614)
medium and rotated on a roller rack at 37°C. As
measured by the Boyden hemagglutination
method, tissue slices and fragments yielded equiv-
alent amounts of antibody, but isolated cells pro-
duced little antibody. Addition of 20% rabbit
serum to the medium increased antibody pro-
duction, but horse, bovine, human, or sheep sera
produced little effect. Antibody production was
maximum between the 6th and 12th hours of in-
cubation, but sometimes continued for 48-72 hr.
The amino acid analogues, y ethyl amino glutamic
acid or p-fluorophenylalanine, inhibited antibody
formation -and these effects were overcome by
glutamic acid or phenylalanine plus tyrosine

AMERICAN ASSOCIATION OF IMMUNOLOGISTS

623

respectively. S;; methionine in the medium was
incorporated into antibody. A net increase in
gamma globulin during incubation was indicated
by isolation of up to twice as much gamma globulin
from the system after incubation as was present
at zero time. The foregoing data indicate that
antibody is synthesized from simple precursors
(amino acids, etc.) in the in vitro system.

2032. Peripheral venous responses to environ-
mental temperature change in man. J. E.
Woop anp J. W. EcksreIn (introduced by E. M.
Fo.LueNsBy). Evans Memorial, Boston Univ.
School of Medicine, Boston, Mass.

Subjects were seated with the forearm inserted
into a two-chamber water plethysmograph. Ex-
ternal hydrostatic pressure exceeded local forearm
venous pressure by 5 mm Hg; thus the effective
local venous pressure (internal minus external
pressure) was reduced to a constant value (1 mm
Hg). Effective local venous pressure was then
increased from 1 to 31 mm Hg by inflating a proxi-
mal cuff. Artefact produced by congestion of the
unpressurized cone of tissue at the proximal end of
the forearm segment volume increase in the distal
chamber only. This volume in cc/100 cc forearm
tissue was called venous distensibility. A high
value was interpreted as relative venodilatation
and a low value as relative venoconstriction. Ten
subjects were studied: in a warm (83°-88°F) and
subsequently a cool (64°-69°F) environment, or
vice versa, on 16 occasions. Six studies were car-
ried out with plethysmographic blood flow meas-
urements in the opposite forearm. Venous dis-
tensibility averaged 33% less (ranging from 12 to
56%) in the cool environment in 14 experiments.
Two subjects had no response to temperature
change. Concomitant blood flow measurements
indicated that the rate of venoconstriction was
much slower than the rate of arteriolar constric-
tion on cooling. However, these rates were similar
on warming. Reduction of plethysmographic water
temperature did not produce venoconstriction.

2033. Comparison between extracellular prod-
ucts of toxigenic and nontoxigenic diph-
theria bacilli. M. Yonepa* anv A. M. Pap-
PENHEIMER, JR. Dept. of Microbiology, New
York Univ. College of Medicine, New York City.
Coproporphyrin III and toxin are released into

the culture medium by toxigenic, lysogenic strains

of C. diptheriae only when growth occurs in the
absence of an exogenous iron supply. We have
compared, under these conditions, growth, total
protein release, toxin, porphyrin and extracellular
nucleic acid production by the sensitive, non-
toxigenic C7 strain and the lysogenic, toxigenic

C7(8) strain derived from C7 by infection with

8-bacteriophage. Bacteria, harvested during the

exponential growth phase, were centrifuged and
washed with deferrated medium. They were then

624

suspended in iron-free medium (to about 0.5 mg
bacterial N/ml) at 32°-34°C with shaking. Growth
continued 6-8 hr. at an ever-declining rate to an
optical density equivalent to about 1 mg bacterial
N/ml. Both strains released about the same
amount of total TCA precipitable protein and of
porphyrin, at a linear rate, during this period, but
only the C7(8) culture liberated toxin. Of the
total protein released by the lysogenic strain, 60-
80% was toxin. No appreciable extracellular
nucleic acid was released until growth, porphyrin
and toxin production had ceased. The nontoxic
protein liberated by C7 had nearly the same solu-
bility and electrophoretic mobility as toxin. How-
ever, the C7 protein does not react with diphtheria
antitoxin nor does it appear to be antigenic for
rabbits.

2034. Thermal inactivation studies with dif-
ferent strains of poliovirus. J. 8S. YOUNGNER.
Virus Research Lab., Univ. of Pittsburgh School
of Medicine, Pittsburgh, Pa.

FEDERATION PROCEEDINGS

Volume 1§

Rate of destruction of infectivity at 50°C was
studied using 19 strains of the 3 immunologic
types of poliovirus; these included 8 type 1 strains,
6 type 2 strains, and 5 type 3 strains. Titered tissue
culture fluids of each strain were adjusted to px
8.3-8.4 and 10-ml aliquots were heated for different
intervals in a water bath. All assays of infectivity
were performed by the plaque count method using
monolayer cultures of trypsin-dispersed monkey
kidney cells. Strain within each type were found
to differ in their thermostability. During the first
hour of heating at 50°C, type 3 strains tended to
be destroyed most rapidly. Mean per cent in-
fectivity remaining at the end of 1 hr. was 25%,
14%, and 0.1% for type 1, 2, and 3 strains, respec-
tively. With continued heating, the rate of in-
activation of type 2 and 3 strains was lower than
in the first hour; this effect was less evident for
type 1 strains. These findings suggest the existence
of poliovirus particles with different degrees of
thermostability at 50°C.

ume 15

C was
ologic
trains,
tissue
to pH
ferent
tivity
| using
onkey
found
e first
Jed to
nt in-
| 25%,
espec-
of in-
r than
nt for
stence
ees of

AUTHOR INDEX

Roman figures refer to serial number of abstract, FEDERATION Pro-
CEEDINGS, Part I; bold figures refer. to serial number of paper in pro-
gram, FEDERATION PROCEEDINGS, Part II. S refers to symposia and special
sessions; P refers to panels; JS refers to Joint Session; and MP, to Motion

Pictures, Page 628.

Abbott, L. D. Jr., 674, 1180
Abeles, R."H., 675, 815
Abernathy, R. S., 1890, 2039
Abood, L. G., 634, 378
Aboody, R., 838, 1365
Abrams, R., 711, 1119
Abramsky, M., 676, 929
Abreu, B. E., 1387, 1523
Ackerman, C. J., 1758, 1891
Ackerman, N. B., 1, 456
Ackermann, W. W., 1891, 1967; 1934, 2009
Adachi, R., 1157, 1900
Adam, D. J. D., 1884, 1852
Adams, E., 677, 1360
Adams, E. C., Jr. 678, 1264
Adams, J. Q., 2, 620
Adams, R., 67, 552
Adams, R. D., 2023, 2049
Adams, W. E., 465, 302
Adamson, D. M., 160, 333
Adelman, W. J., 661, 247
Adelstein, S. J., 1639, 831
Adeyemo, A., 1569, 1408
Adler, F. L., 1892, 2072
Adler, T. K., 1281, 1497
Adolph, E. F., 404S
Aebi, H., 1087, 1250
Aftergood, L. 1759, 11
Agee, J. 1270, 115
Agersborg, H. P., Jr., 3, 720; 27, 731
Agre, K., 1282, 1460
Agulnek, M., 228, 522
Ahiness, P., 172, 630
Ahiquist, R. P., 1490, 1564
Ainis, H., 324, 126
Aisen, P., 992, 622
Ajl, 8. J., 1893, 2052
Aladjem, F., 679, 1089
Albanese, A. A., 680, 1284
Albrecht, A. M., 681, 1327
Alcaraz, M., 293, 533
Aleocer-Cuaron, C., 292, 763
Aldes, J. H., 1067, 1789
Aldrich, P., 1852, 1890
Aldrich, R. A., 1200, 1159
Alex, T., 1128, 1126
Alexander, H. D., 180S
Alexander, J. C., 1760, 1836
Alexander, W. M., 1387, 1523
Alfin-Slater, R. B., 760, 59; 1759, 11; 1776,
1851; 1783, 1838
Alkjaersig, N., 1151, 40
Alksne, J. F., 4, 332
Allen, F. W., 776, 1362
Allen, G., 1569, 1408
Allen, J. G., 278, 43
Allen, J. R., 1089, 1007
Allen, R. F., 1753, 12
Allensworth, J. W., 306, 282
Allison, J. B., 1761, 1905

Allred, J., 1818, 1897

Alpen, E. L., 139, 669

Alper, C., 1282, 1460

Alpert, N. R., 5, 510

Altamirano, M., 6, 245; 536, 442

Altland, P. D., 7, 511; 69, 628; 1684, 1716;
1721, 1717

Altszuler, N., 8, 312; 637, 311

Alvig, O. H., 988, 1061

Alvord, E. C., 938, 1724

Alworth, B. L., 1628, 1419

Amassian, V. E., 9, 469

Ambrose, A. M., 1288, 1561; 1313, 1667

Ambrus, J. L., 1290, 122

Ames, B. N., 682, 1141

Ames, 8. R., 1809, 1847

Amiraian, K., 1983, 2066

Amrein, M., 411, 671

Anason, A., 81, 369

Andersch, M. A., 683, 125

Anderson, C. E., 1068, 1375

Anderson, D. G., 799, 1028

Anderson, E. I., 1185, 1249

Anderson, E. P., 684, 949

Anderson, F. F., 1284, 1579

Anderson, F. 8., 644, 384

Anderson, H. H., 1435, 1679

Anderson, J. T., 255, 500; 603, 505b; 1762, 3

Anderson, N. G., 10, 1341

Anderson, R. C., 1544, 1645

Anderson, R. 8., 11, 567

Andersson, B., 12,M.P., p. 628

Andose, A., 1585, 1510

Andres, R., 13, 549

Andresen, R., 1640, 1702

Andrew, W., 275S

Andrews, J. C., 1273, 1384

Andrews, J. 8., 179S

Andrus, 8. B., 1763, 8; 1790, 10; 1831, 6

Anfinsen, C. B., Jr.,JS, p. 628; 685, 822

Angerer, C. A., 652, 800

Angevine, D. M., 1661, 1784; 1705, 1767

Anliker, J., 1835, 1802

Annegers, J., 339, 687

Anthony, W. L., 704, 82

Antoniades, H. N., 686, 1271

Antopol, W., 672, 802; 673, 673; 1641, 137

Anzola, J., 513, 281

Aposhian, H. V., 687, 861; 1285, 1663

Appert, H., 212, 611

Aprison, M. H., 14, 325

Aras, Albert, 1652, 1707

Archibald, E. R., 347, 770

Armocida, E., 1115, 1377

Armstrong, M. D., 1155, 1171

Armstrong, W. D., 15, 104; 749, 135

Arnold, A., 1764, 1848; 1766, 1849

Anrich, L., 688, 1833

Arnoff, 8., 689, 927

Arons, W. L., 1, 456

Arquilla, E. R., 1945, 1965
Arroyave, G., 1872, 1837
Arscott, P., 1649, 24

Arscott, P. M., 1642, 39
Artom, C., 690, 1295

Asano, T., 315, 396

Asenjo, C. F., 782, 923
Ashburn, L. L., 1595, 1616a
Asher, D. R., 582, 612

Asher, D. T., 1437, 1640
Ashmore, J., 217, 588; 691, 1063
Ashworth, E., 836, 1359
Assali, N. 8., 844, 406

Aston, R., 1286, 1582
Astrachan, L., 692, 1191
Asuncion, C. L., 408, 539
Atkins, J., 1883, 1916
Auditore, G. V., 1287, 1562; 1428, 1412
Aull, J. C., 354, 661

Auvil, D. K., 674, 1180
Auyong, T., 33, 732

Avery, M., 1372, 1568

Aviado, D. M., Jr., 16, 351
Awapara, J., 791, 1243
Axelrod, A. E., 1859, 1819
Axelrod, B., 693, 829

Axelrod, J., 1288, 1443; 1577, 1440
Ayer, J. P., 1643, 1745

Ayres, N. 8., 184, 659

Azima, H., 17, 323

Baart, N., 1289, 172; 1368, 1689; 1374,
176; 1568, 174

Bacchus, H., 18, 454

Bach y Rita, P., 1454, 1652

Bachhawat, B. K., 694, 1068

Back, N., 1290, 122

Bacon, J. A., 1857, 1880

Baeder, D. H., 1291, 88; 1566, 87

Baer, J. E., 668, 701

Baetjer, A. M., 326, 729

Baez, 8., 576, 675; 1292, 1664

Baggett, B., 1134, 1160

Bahn, R. C., 252, 280

Baily, N. A., 445, 690

Bain, J. A., 1293, 1516

Baird, C. D. C., 1787, 181%; 1844, 1827

Baird, H. L., 98, 153

Baker, B. L., 20, 730

Baker, D. G., 274, 586

Baker, G. D., 704, 82

Baker, R. D., 541, 704; 1644, 1715

Baker, W. W., 572, 270; 1294, 1665

Bakshy, 8., 516, 202

Baldes, E. J., 295, 336

Baldridge, H. D., 1604, 187

Bale, W. F., 569, 2003; 1645, 1758

Baléa, T., 126, 742

Balek, R. W., 1295, 1591

Balis, M. E., 695, 1263

Balke, B., 21, 512; 179, 681; 643, 680

Ball, E. G., 995S

Ball, W. C., Jr., 22, 214; 252, 280

Ballard, A., 1631, 1426

Balourdas, T. A., 23, 387

Baltscheffsky, M., 1170, 1019

Banks, J., 1258, 897

Bannett, S. M., 644, 384

Baptist, J. N., 1227, 832

Barban, S., 696, 54

Barboriak, J. J., 1765, 1820

Barclay, R. K., 859, 1034

Bardhanabaedya, S., 145, 745

Barger, A. C., 24, 502

Barker, H. A., 697, 941

Barker, J. N., 25, 509

Barker, P., 1706, 1781

Barker, 8. B., 26, 461; 1562, 1512

Barlow, G., 3, 720; 27, 731

Barlow, J. L., 2020, 1186

Barner, H., 864, 1117

Barnes, T. C., 1296, 1532

Barnett, R. C., 28, 718

Barnum, C. P., 918, 982

Barraclough, C. A., 29, 240

Barrett, W. E., 30, 517

Barrows, C. H., 277S

Barry, G. T., 1556

Bartholomew, R. A., 901, 1791

Bartlett, P. D., 698, 1076

Bartlett, R. G., 31, 242

Bartlett, R. G., Jr., 377, 772

Barton, A. D., 32, 980

Barton, B., 527, 436

Barton, E., 679, 1089

Bascom, W. D., 507, 593

Bascoy, L. T., 816, 100

Basek, M., 200, 309

Basford, R. E., 699, 1232

Basinski, D. H., 1924, 1981

Bass, A. D., 1342, 1056; 1362, 1618

Bassett, C. F., 1877, 1807

Bastian, J. W., 1297, 121; 1425, 150

Bateman, J. B., 170, 1105

Bates, R. W., 761, 1326

Batterman, R. C., 1298, 1621; 1396, 1622;
1406, 1538

Battersby, W. 8., 461, 417

Batts, A. A., 609, 525

Baughan, M. A., 1884, 1852

Baum, §. J., 1299, 29

Baumann, C. A., 1791, 1801

Baumann, D., 1565, 1449

Baumgardt, J. P., 33, 732

Baxter, C. F., 700, 1021

Baxter, J. 1309, 1705

Baxter, J. H., 1300, 1755

Bay, E., 34, 1493

Bay, I., 34, 1493

Beach, V. L., 367, 235

Bean, J. W., 35, 689

Beardmore, W. B., 1993, 1953

Bearn, A. G., 36, 218

Beaton, G. H., 701, 194

Beatty, C. H., 37, 709

Bechtel, A. A., 38, 359

Beck, L. V., 39, 452

Beck, W. 8., 821, 1086

Becker, E. L., 1894, 2062; 1906, 2064; 1974,
2021

Becker, J. R., 1712, 1711

Becker, R. R., 1122, 955; 1193, 1037

Becker, W. W., 975, 1370

FEDERATION PROCEEDINGS

Beecher, H. K., 1301, 1495; 1401, 1496

Beers, R. F., Jr., 40, 1259

Beerstecher, E., Jr., 702, 863

Begany, A. J., 1302, 1657

Begg, R. W., 703, 50

Beher, W. T., 704, 82

Behnke, A. R., 41, 548

Behnke, A. R., Jr., 184, 659

Behrmann, V. G., 42, 575

Beidler, L. M., 43, 605; 613, 604

Beiler, M. J., 705, 1790

Beinert, H., 706, 1214

Beisaw, N. E., 46, 594

Belford, J., 1303, 1461

Bell, F. E., 707, 893

Bell, F. K., 1602, 1514

Bell, J. B., 44, 733

Belleville, R. E., 1377, 1505

Bellin, E. L., 1101, 55

Benaron, H. B. W., 299, 374

Bender, C. H., 1371, 1584

Bender, M. B., 461, 417; 632, 535

Benditt, E. P., 1646, 1792

Benerito, R. R., 708, 1847

Benesch, R., 709, 972

Benesch, R. E., 709, 972

Bennett, A. L., 1613, 1698

Bennett, B. L., 1992, 1950

Bennett, H. 8., 4, 332

Bennett, L. L., Jr., 710, 53

Bennett, L. R., 1648, 136; 1694, 130

Bennett, M. J., 1839, 1935

Benson, W. M., 1304, 1625

Bentley, M., 711, 1119

Bentley, R., 712, 1218

Benzinger, T. H., 45, 635; 425, 636

Berg, C. P., 756, 1125

Berg, P., 713, 1078

Bergen, J. R., 46, 594

Berger, C. R. A., 883, 1302

Berger, E., 570, 110

Berger, F. M., 1305, MP, p. 628; 1419,
1660

Berger, H., 392, 624

Berglund, E., 47, 388

Berglund, F., 118, 295

Berliner, D., 518, 1356

Berliner, D. L., 714, 1351

Berke, H. L., 48, 717

Berkowitz, E. C., 49, 531

Berlin, L., 50, 734

Berman, H. J., 51, M.P., p. 628; 52, 735

Berman, I., 53, 314

Berman, M. D., 546, 252

Bermes, E. W., Jr., 715, 1361

Berne, R. M., 54, 389; 1630, 1486

Bernfeld, P., 716, 1346

Bernheimer, A. W., 1989, 2057

Bernstein, H., 55, 458

Bernstein, W., 720, 1336

Berthet, J., 1091, 1155

Berthet, L., 881, 1073

Berry, J. F., 1020, 1004

Berueffy, R. R., 1441, MP, p. 628

Bessman, M. J., 717, 1344

Bessman, §. P., 968, 1148

Best, C. H., 519, 712

Bethell, F. H., 1740, 46

Beutner, E. H., 1306, 1554

Beutner, R. H., 56, 185; 57, 736

Bevans, M., 1647, 1778; 1904, 1910

Beyler, A. L., 1764, 1848; 1766, 1849

Bhamarapravati, N., 1668, 1759

Bhargava, K. P., 1307, 1569

Volume 1§

Biale, J. B., 676, 929

Bialy, J. J., 1238, 1379
Bianconi, A. M., 39, 452

Bier, M., 1210, 887

Bieri, J. G., 1767, 1850
Bierman, H. R., 1449, 626
Billig, D. M., 58, 206
Billings, M. S., 1648, 136
Binion, J. T., 59, 210; 432, 211
Birch, J. H., 1535, 1531

Bird, E., 1793, 1845
Birnbaum, 8. M., 718, 1808
Birzis, L., 60, 637

Bischoff, F., 719, 658

Bittner, J. J., 107, 644
Bjerknes, C., 720, 1336
Bjorklund, B., 885, 1957
Black, F. L., 1895, 1954
Black, J., 251, 45

Black, S., 721, 1144
Blackmore, W. P., 1308, 1587
Blair, J. R., 658, 629

Blake, W. D., 61, 501

Blatt, I. M., 1940, 2027

Blau, M., 1896, 2001

Bliss, C. I., 1421S

Bloch, A., 1513, 90

Bloch, E., 722, 1070

Bloch, E. H., 1897, MP, p. 628
Bloch, K., 1056, 847

Blockus, L. E., 1369, 1427
Bloodworth, Jr., J. M. B., 1649, 24
Bloom, B. M., 723, 1158
Blum, H. F., 62, 698

Blum, J. J., 63, 261; 68, 257
Blumberg, H., 1309, 1593; 1310, 1666
Blumenfeld, S., 529, 788
Blumenstein, J., 1257, 1742
Bocek, R. M., 37, 709
Bocklage, B. C., 1111, 1168
Bodenlos, L. J., 1747, 1765
Bodi, T., 64, 737

Boell, E. J., 400S

Boeri, E., 751, 1236

Bogart, R., 369, 645
Bogdanski, D. F., 1311, 1634; 1356, 1636;

1605, 1635
Boggiano, E. M., 1169, 1247
Bogoch, 8., 826, 996
Bohner, B., 1329, 1668
Bohner, H. J., 1965, 2070
Bohr, V. C., 31, 242
Bolene-Williams, C., 342, 769
Bollman, J. L., 823, 1821; 1650, 1703;
1701, 1762
Bond, V. P., 65, 106; 500, 111a
Bonnycastle, D. D., 1312, 1548
Booker, W. M., 1499, 1417; 1569, 1408
Booth, A. N., 724, 1274; 1313, 1667
Borison, H. L., 66, 108; 1307, 1569
Bornstein, A. M., 148, 610
Boroff, D. A., 288, 666; 1898, 2051
Borsook, H., 1869S
Borst, H., 47, 388
Boryezka, A., 816, 100
Borzelleca, J. F., 1314, 1462
Boss, W. R., 238, 758
Bothelho, 8., 67, 552
Bott, P. A., 725, 294
Boucek, R. J., 337, 1201; 1507, 91
Bouchard, B. 8., 1827, 1932
Bourke, A. R., 1315, 1404; 1373, 1405
Bowen, W. J., 68, 257
Bowman, H. §., 1537, 1691

Bro

Bro
Bro
Bro
Brov
Broy
Broy
Broy

lume 1§

150, 1708;

, 1408
667

, 1405

March 1956

Boyd, C. E., 1316, 138a

Boyd, E. M., 1316, 133a

Boyd, L. J., 235, 757

Boyd, R. B., 101, 740a

Boyer, G. S., 1920, 1942

Boyer, P. D., 726, 1013

Bozeman, F. M., 1899, 1988; 1995, 1987

Brace, K. C., 69, 623

Bracken, E. C., 1651, 1722; 1717, 1721B

Bradford, W. L., 1753, 12

Bradley, A. F., 590, 682

Bradley, A. J., 1312, 1543

Bradley, S. E., 645, 330

Bradlow, H. L., 727, 1161

Brady, A. J., 70, 395

Brady, R. O., 1129, 1145

Bragg, A. D., 1615, 1560

Braham, J. E., 1872, 1837

Bramante, P. O., 209, 335

Brand, L., 1482, 1389

Brandfonbrener, M., 556, 198; 615, 355

Brannick, L. J., 30, 517

Brauer, R. W., 1317, 1650

Braunschweig, L. W., 668, 701

Braunwald, E., 71, 381; 99, 380;
214, 568; 577, 288

Brecher, G. A., 543, 674

Brecht, E. A., 1068, 1875

Brehme, A. C., 173, 708

Brendel, R. R., 705, 1790

Brennan, M. J., 1924, 1981

Bressani, R., 1768, 1860

Brewer, W. D., 1769, 1909

Briggs, G. M., 1767, 1850

Bright, H. B., 1810, 1835

Brin, M., 728, 1282

Brink, F., 315, 396

Brinkhous, K. M., 1746, 113

Briscoe, S. M., 72, 656

Briscoe, W. A., 73, 308

Britton, B., 1254, 1065

Brodie, B., 1465, 1630

Brodie, B. B., 1356, 1636; 1376, 1393; 1415,
1638; 1423, 1631; 1482, 1389; 1561, 1643;
1573, 1629

Brodsky, W. A., 74, 341

Brody, A. W., 75, 224

Brody, 8., 136, 626; 329, 766

Bromley, J. W., 1770, 1857

Bronk, D. W., 121, 253

Bronner, F., 729, 1181; 1880, 1925

Brooks, C. McC., 592, 484

Brooks, P. M., 76, 134; 229, 105

Brooks, R. H., 718, 1808

Brooks, V. B., 77, 415

Broquist, H. P., 730, 1306

Bross, I., 1404, 1555

Brown, A., 1652, 1707

Brown, E. B., Jr., 78, 685

Brown, E. 8., 79, 233

Brown, F. C., 731, 1146

Brown, G. B., 871S

Brown, G. C., 1900, 1947

Brown, G. W., 117, 326

Brown, I. W., Jr., 167, 334

Brown, J. H. U., 80, 738; 81, 369

Brown, J. R., 1089, 1007

Brown, M. L., 117, 326

Brown, R. K., 732, 1112; 1952, 2023

Brown, R. R., 733, 1176; 1079, 1173

Brown, R. V., 1318, 1434

Brown, T. G., Jr., 1319, 213

Brownell, L. E., 178S, 742, 1809

Brownell, L. W., 1142, 16

AUTHOR INDEX

Brozek, J., 428, 505a

Bruce, C., 1716, 1772

Bruce, W. F., 1302, 1657
Bruemmer, J. H., 1847, 1822
Bruemmer, N. C., 1212, 1345
Bruhn, J. M., 182, 265
Brummond, D. O., 734, 1255
Brunder, J., 1707, 1777
Bruner, H. D., 1572, 1488
Brunish, R., 735, 1193
Bruno, D., 1858, 7

Brush, M. K., 1761, 1905
Brust, M., 524, 787
Brust-Carmona, H., 292, 763
Bubl, E. C., 1838S

Buch, F. G., 82, 739
Buchanan, D. L., 736, 1041
Buchanan, G. D., 83, 414
Buchanan, J. M., 993, 913
Buchwald, N. A., 84, 328
Buckley, S. M., 1653, 1718
Budovich, T., 1063, 952
Bueding, E., 1320, 1474
Buehler, H. J., 1048, 844
Buescher, E. L., 1996, 1992
Buettner, K. J. K., 85, 726
Bulaschenko, H., 611, 592
Bull, F. E., 1654, 1786
Bullard, R. W., 86, 477
Bumpus, F. M., 737, 557
Bunker, J. P., 1421, 1515
Burbridge, T. N., 1424, 1480
Burch, H. B., 738, 1281
Burchenal, J. H., 1780, 17 ©
Burge, G. J., 136, 628
Burger, M., 1171, 1134
Burgess, L. E., 87, 1824
Burgison, R. M., 1321, 1599
Burk, D., 739, 933; 1267, 99
Burke, J. C., 1943, 1980
Burke, W. T., 740, 886
Burkhalter, A., 1322, 171
Burma, D. P., 741, 857
Burman, J., 1094, 1207
Burns, C. H., 178S; 742, 1809
Burns, J. J., 1323, 954; 1482, 1389
Burns, M. J., 1778, 14

Burr, W. W., Jr., 1255, 808; 1771, 1846
Burrill, L., 1769, 1909

Burris, R. H., 741, 857
Burrows, B. A., 1982, 1983
Burstein, S., 743, 1071; 1711, 1727
Burstein, B., 143, 329
Burton, A. C., 218, 634
Burton, R. B., 912, 1350
Busch, H., 744, 1476

Bush, M. T., 1324, 1388
Buskirk, E., 428, 505a; 603, 505b
Buskirk, E. R., 88, 506; 316, 476
Butler, T. C., 1325, 1387
Butler, W. M., Jr., 1503, 1452; 1529, 1453
Butts, J. 8., 1838S

Buxbaum, J. D., 89, 371
Buznitsky, A., 2007, 2045
Byerrum, R. U., 780, 1127
Byron, J. W., 1290, 122

Cadmus, R. A., 1400, 1418; 1574, 1535
Cafruny, E., 1371, 1584

Cahill, G. F., Jr., 691, 1063

Calhoon, T. B., 90, 392

Calhoun, D. W., 1422, 1620

ili

Callow, A. D., 1509, 19
Calloway, D. H., 1772, 1844
Calvary, E., 278, 43
Cameron, A., 1326, 1522
Cammermeyer, J., 656, 420
Campbell, A. H., Jr., 1400, 1418; 1574,
1535
Campbell, B., 91, 740
Campbell, D. C., 1654, 1786
Campbell, D. H., 679, 1089; 1917, 2025
Campbell, E. W., 92, 664
Campbell, J., 102, 523
Campbell, J. A., 1842, 1840
Canahuati, 8. M., 1587, 1644
Cander, L., 67, 552; 93, 356
Canellakis, E. 8., 745, 1116
Cantarow, A., 94, 1260; 460, 408
Cantoni, G. L., 746, 1245
Cantrell, W., 1327, 1548
Canzanelli, A., 95, 111
Caputto, R., 1013, 1277
Carbon, J. A., 1278, 888
Carlat, L., 906, 986
Carleton, A., 19; 576, 675
Carlson, G. L., 1022, 1246
Carlson, L. D., 130, 537; 462, 262
Carlsson, A., 1573, 1629
Carnes, W. H., 1655, 1749
Carpenter, E., 1864, 1936
Carpenter, F. G., 96, 249
Carpenter, F. H., 747, 1109
Carpenter, H. M., 339, 687
Carpenter, K. J., 1773, 1806
Carr, C. J., 1845, 1451
Carroll, K. K., 97, 5
Carroll, R. T., 64, 737; 98, 153
Carson, 8., 1310, 1666
Carter, C. E., 748, 839
Carter, H. E., 868S
Carter, Y., 1656, 1726; 1690, 1725
Cartwright, G. E., 1801, 1887
Case, J. D., 1657, 1712
Case, R. B., 71, 381; 99, 380; 577, 288;
586, 212
Casselman, W. G. B., 537, 368; 1092, 447
Cassin, 8., 100, 581
Caster, W. O., 15, 104; 749, 135
Castiglioni, G., 1115, 1377; 1225, 1780
Castro-Mendoza, H., 821, 1086
Catchpole, H. R., 555, 2026
Cavalieri, L. F., 750, 1048
Cavenaugh, D. J., 877, 931
Cavert, H. M., 101, 740a
Celander, D. R., 948, 118
Cerceo, E., 2010, 826
Chace, J. A., 149, 553
Chaikof, L., 102, 523
Chaikoff, I. L., 357, 446; 1856, 4
Chakales, H. H., 142, 652
Chaloupka, M., 1774, 1815
Chambers, A. H., 103, 602
Chance, B., 751, 1236
Chang, C. C., 752, 1210
Chang, M. C., 104, 485
Chang, M. L. W., 1775, 1811
Chapin, J. L., 105, 679
Chargaff, E., 1153, 1051
Charipper, H. A., 82, 739
Charles, M. L., 1328, 1482
Chart, J. J., 106, 516
Chase, M. W., 2029S
Chaudhry, A. P., 107, 644
Cheek, D. B., 108, 397
Cheek, W. R., 268, 366

Cheever, F. S., 1901, 158

Cheldelin, V. H., 940, 1238; 943, 907

Chen, G., 1329, 1668; 1330, 1432

Chen, J. Y. P., 1831, 498; 1332, 1669

Chen, K. K., 1545, 1396

Chen, 8S. D., 1870, 1903

Chen, Y. M., 760, 59

Cheng, A. L. 8., 1776, 1851

Cheng, C. 8., 329, 766

Cheng, C. W., 753, 1199

Cherbakoff, A., 109, 493

Chernick, 8. 8., 754, 1278

Chernish, S. M., 1333, 1464

Chervenka, C. H., 1256, 1111

Chessin, M., 1334, 1637

Child, G. P., 1335, 1647

Childs, A. W., 120, 451; 645, 330

Chin, P. H., 2000, 162

Chinard, F. P., 755, 215

Chiodi, H., 110, 513

Chodos, R. B., 1123, 1322

Choitz, H. C., 756, 1125

Chow, B. F., 779, 1787; 1814, 1826; 1850,
1823

Christensen, E. K., 1600, 1697

Christensen, H. N., 1104, 1137

Christensen, J., 1614, 1699; 1336, 1487

Christian, H. H., 1337, 1536; 1479, 1534

Christian, J. J., 1658, 1733

Christman, A. A., 768, 921

Christy, N. P., 323, 405

Chu, L. W., 1903, 1955

Chu, M. W., 1272, 1383

Chusid, J. G., 111, 741

Ciesielski, J., 504, 784

Clark, D. A., 757, 1074

Clark, G. M., 1596, 1409

Clark, H., 1777, 1862

Clark, J. W., 628, 28

Clark, J. W., Jr., 1657, 1712

Clark, R. T., Jr., 112, 584; 622, 586; 650,
585

Clark, 8. S., 87, 1824

Clark, W. G., 472, 653

Clark, W. H., Jr., 1659, 1769

Clarke, D. W., 758, 905

Clarke, G. E., 244, 33

Clarke, N. P., 113, 505; 671, 504

Clasen, R., 114, 272

Clayton, C. C., 759, 93

Clegg, K. M., 1773, 1806

Clements, G. R., 1660, 1763

Clements, J. A., 115, 232

Clements, M. M., 226, 390

Clemmons, J. J., 1661, 1784

Clifton, K H., 1662, 127

Cline, R. E., 818, 1114

Clouet, D. H., 1057, 998

Cobb, L. A., Jr., 371, 207

Cobb, P., 411, 671

Coburn, A. F., 1902, 2055

Cochin, J., 1288, 1443

Cochran, D. G., 517, 1152

Cochran, K. W., 1903, 1955

Code, C. F., 116, 606; 427, 607

Coffin, D. L., 1954, 1999

Cohen, B. D., 117, 326

Cohen, E., 933, 895

Cohen, J. J., 118, 295

Cohen, L. A., 119, 421

Cohen, P. P., 869, 1298

Cohen, 8. I., 1904, 1910

Cohen, 8. 8., 864, 1117

Cohn, D. V., 1338, 1222

FEDERATION PROCEEDINGS

Cohn, G. L., 1018, 881

Cohn, 8. H., 1339, 107; 1794, 30
Cole, L. J., 1340, 71

Coleman, R. D., 760, 59
Collazos, C., 1883, 1916

Collins, D. M., 306, 282

Collins, V. P., 1531, 76
Colowick, 8. P., 934, 987
Comar, C. L., 1881, 1865
Combes, B., 120, 451; 645, 330
Combs, A. M., 738, 1281
Condliffe, P. G., 761, 1326
Congdon, C. C., 419, 165
Coniglio, J. G., 762, 1288
Conley, C. L., 322, 155

Conn, E. E., 763, 928

Conn, H. L., Jr., 1520, 1471
Connelly, C. M., 121, 253
Conner, P. K., Jr., 1341, 1604
Conrad, E., 1342, 1056

Conrad, M. C., 422, 465

Conroy, C., 1343, 1524

Conte, F. P., 1663, 1793
Contopoulos, A.'N., 122, 521
Conwell, D. P., 1914, 2033
Conzelman, G. M., Jr., 1344, 1670
Cook, E. B., 1604, 187

Cook, J. L., 764, 818

Cook, J. 8., 62, 698

Cooke, P., 114, 272

Coon, J. M., 1547, 190

Coon, M. J., 694, 1068

Cooper, C., 765, 1015

Cooper, D. Y., 123, 300

Cooper, J. A. D., 124, 217
Cooper, T., 125, 650

Copeland, D. H., 1778, 14
Copeland, J. E., 1541, 1401
Copley, A. L., 126, 742
Corcoran, A. C., 409, 566; 1354, 1583
Corcoran, J. W., 766, 1354
Cordes, F. L., 1449, 626
Cordoba, F., 885, 1957

Cormier, M. J., 127, 642; 509, 814
Cornwell, D. G., 365, 1348; 853, 84
Corristan, E. C., 1905, 1994
Corson, E. O’L., 128, 350
Corson, 8. A., 128, 350

Corwin, L. M., 1664, 1289
Cosmides, G. J., 1345, 1451
Costa, E., 202, 711; 203, 755
Costa, F., 816, 100

Cotlar, A. M., 279, 760

Cotlove, E., 129, 443

Cotten, M. deV., 1319, 213; 1346, 284
Cottle, W., 130, 537

Cotzias, G. C., 1725, 193

Couch, J. R., 1099, 1885; 1789, 1872
Coulomb, B., 1169, 1247
Coulson, R. A., 291, 762; 769, 437
Cournand, A., 73, 308; 214, 568; 645, 330
Couves, C. M., 1347, 1581
Covey, B. M., 163, 713

Cowan, C. R., 1092, 447
Cowgill, R. W., 770, 1177

Cox, G., 1737, 1776

Cox, R. P., 1783, 1838

Coxe, W. 8., 131, 266

Craig, J. M., 1665, 1760

Craig, J. W., 132, 196

Cramer, J. W., 1779, 1927
Crampton, C. F., 771, 1045
Crandall, D. I., 772, 1172
Crandall, E., 133, 743

Volume 1§

Crane, F. L,, 854, 1281
Crawford, E. 8., 1347, 1581
Crescitelli, F., 134, 526
Crespi, H., 984, 966

Cress, E. A., 961, 109
Crokaert, R., 773, 1299
Cronheim, G., 1348, 1683
Cronkite, E. P., 500, 111a
Crotty, W. J., 135, 643
Crout, J. R., 1689, 1740
Crowley, W. P., Jr., 207, 570
Cuajunco, F., Jr., 620, 497
Cuchie, F. T., 1349, 1580
Cuckler, A. C., 1350, 1546
Cueto, C., Jr., 1363
Cunningham, D. J., 1304, 1625
Curry, D. M., 701, 194
Curtis, J. M., 1222, 1649
Cushman, W. F., 1906, 2064

Dackset, L. J., 1048, 844
DaCosta, F. M., 1499, 1417
Dagg, C. P., 774, 95
Dagg, M. K., 1780, 17
Dahl, L. K., 1666, 1735
Dahl, N., 523, 370
Dalby, A., 1029, 119
Dale, H. E., 136, 628
Dallam, R. D., 241, 204
Dam, R., 1840, 1886
Danauskas, J. X., 1899, 1988
D’Angelo, S. A., 137, 744
Daniel, J. W., 1667, 935
Daniel, L. J., 1021, 168
Daniels, M., 793, 854
Darby, T. D., 1351, 289
Darby, W. J., 1830, 1933
D’Asaro, B. 8., 1352, 1486
Dattilo, J. V., 1670, 1710
Daughaday, W. H., 775, 1357
Davenport, F. M., 1907, 2014; 1930, 2013
Davenport, H. W., 138, 608
David, N. A., 1523, 1690
Davidsohn, I., 2007, 2045; 1908, 2046
Davidson, A. G., 829, 84
Davidson, C. 8., 728, 1282
Davidson, D. G., 452, 293
Davidson, I. W. F., 519, 712
Davidson, J. D., 1577, 1440
Davies, D. D., 958, 122
Davin, J. C., 1539, 1463; 1597, 1537
Davis, A. K., 139, 669
Davis, D. L., 1853, 173
Davis, F. F., 776, 1862
Davis, G. D., 140, 419
Davis, H. L., 141, 306
Davis, J., 906, 986
Davis, J. E., 1403, 1675
Davis, J. H., 777, 1363
Davis, J. O., 22, 214; 252, 280
Davis, J. R., 5, 510
Davis, J. W., 778, 1241
Davis, L., 390, T14
Davis, O. F., 1510, 68
Davis, R. L., 779, 1787
Dawe, A. R., 373, 546
Day, E. D., 1896, 2001
Day, E. J., 180S
Day, P. L., 795, 164; 1781, 1873; 1784,
1828; 1869, 1875; 1885, 1895
Dayton, H. B., 1309, 1593
Deal, C. P., Jr., 142, 652
Deane, H. W., 204, 527

ee ee ee ee oe ee ee ee - -

~B-B-B- BB h-hh h-hh eo hoo hohohohohoRoRcRechch MRE none ne eee eee ee eee

lume 1§

930, 2013

133 1784,

March 1956

DeBakey, M. E., 1347, 1581

de Beer, E. J., 1508, 1655; 1826, 1894

de Bernard, B., 699, 1232

de Bodo, R. C., 8, 312; 637, 311

DeBoer, B., 1448, 1513

Debons, A. F., 18, 454

de Bruin, C. H., 1200, 1159

deCastro, F. T., 1259, 1381

Decibus, F., 1513, 90

Decker, C. F., 780, 1127

Decker, L. E., 781, 1128

DeEds, F., 724, 1274; 1313, 1667; 1627,
1397

De Giovanni, R., 1276, 1183

de Groot, J., 258, 416

de Gubareff, T., 1385, 1457

Delafield, F., 1130, 1206

del Campillo, A., 782, 923

De Ley, J., 794, 950

Delgado, J. M. R., 143, 329

del Greco, F., 1354, 1583

DeLong, C. W., 1191, 1307

DeLong, D. C., 1777, 1862; 1782, 1864

De Luca, H. F., 788, 1280

deMaar, E. W. J., 1355, 1571

De Meio, R. H., 784, 1066

Dement, W., 144, 418

DeMeyers, M., 1333, 1464

Deming, Q., 1356, 1636

DeMoss, J. A., 785, 875

Denison, A. B., Jr., 145, 745; 146, 337;
571, 433

Denison, M. E., 147, 450

Dennis, E., 1455, 1603

Dennis, W. H., 148, 610

Denslow, J. S., 149, 553

Depalma, R. E., 1140, 862

Depocas, F., 150, 540

Derby, M. B., 1829, 1917

De Ritter, E., 1833, 1877

De Salva, S. J., 1357, 1533

Despopoulos, A., 151, 292

Dessauer, H. C., 786, 968

Detrick, L. E., 1358, 1615

Deuel, H. J., Jr., 760, 59; 1759, 11; 1776,
1851; 1783, 1838

Deutsch, A., 787, 1213

Deutsch, H. F., 788, 1194

Devi, A., 1278, 888

DeVito, J. L., 152, 423

Dewey, K. F., 1139, 1197

Dewey, W., 569, 2003

Dexter, L., 371, 207

Deyrup, I., 153, 439

Diao, E., 1819, 1922

Dick, E., 1870, 1903

Dickman, S. R., 789, 1475

Dickos, J. F., 1901, 158

Didisheim, P., 154, 151

Dieckert, J. W., 1100, 1294

Dietrich, L. 8., 1078, 885

Di Ferrante, N., 1102, 1309

Dill, W. A., 155, 746; 237, 1478

Dille, J. M., 1395, 1651

Diller, E. R., 951, 855

Di Luzio, N. R., 156, 668; 157, 747

DiNella, R. R., 158, 663

Dinning, J. S., 1781, 1873; 1784, 1828;
1869, 1875

DiPalma, J. R., 159, 488

Dische, Z., 790, 948

Diserens, H. W., 451, 208

DiStefano, V., 1359, 1573

Dixon, F. J., 2031S

AUTHOR INDEX

Dobson, E. L., 160, 333
Doctor, V. M., 791, 1243
Doezi, J., 792, 236
Dodson, G., 905, 1242
Dodson, M. J., 674, 1180
Doggett, M. C., 1430, 1578
Dohan, F. C., 611, 592
Doherty, D. G., 1154, 891
Doisy, E. A., 1111, 1168
Doisy, E. A., Jr., 793, 854
Domanski, T. J., 161, 514
Domino, E. F., 1442, 1431
Domino, E. P., 1330, 1432
Domizi, D. B., 465, 302
Donaldson, D. M., 1959, 157
Dorfman, R. I., 722, 1070; 723, 1158;
1134, 1160
Doty, R. W., 514, 467
Doudney, C. O., 162, 70
Doudoroff, M., 794, 950
Dougherty, T. F., 298, 148
Doughty, S., 1407, 1465
Douglas, D. E., 846, 1477
Douglas, J., 999, 816
Douglass, C. D., 795, 164
Doull, J., 1360, 273 1411, 1677
Dounce, A. L., 1075, 838
Drabkin, D. L., 1006, 1283
Dratz, A. F., 163, 713
Dreifus, L., 579, 503
Dreisbach, R. H., 1361, 1671
Drill, V. A., 1422, 1620; 1592, 1654
Drisko, R. W., 1135, 820
Driver, R. L., 1412, 1606
Drobeck, H. P., 1967, 1948
Drucker, W. R., 132, 196
Drummond, G. I., 1189, 1082
Druskin, M. 8., 228, 522
Drutz, E., 1674, 1752
Dryer, R. L., 796, 1335
D’Souza, P. B., 1535, 1531
Dubin, A., 1716, 1772
Dubnick, B., 1334, 1637
Dubnoff, J., 679, 1089
DuBois, K. P., 1504, 1688; 1518, 74
Dubuc, J., 1163, 1217
Duff, F., 47, 388
Duggan, E. L., 797, 1049
Dulit, E., 829, 846
Dumm, M. E., 164, 203; 480, 299
Dunlap, M. K., 1459, 1681
Dunn, C. E., 1362, 1618
Dunnebacke, T. H., 798, 1971
Dimnenberger, M., 802, 1353
Durbin, R. P., 887, 614
Durell, J., 746, 1245; 799, 1028
Durham, W. F., 1363, 1407
Dury, A., 165, 58; 166, 748
Dury, M., 166, 748
Duvall, R. C., 779, 1787
Dwyer, F. M., 551, 78
Dyer, A. E., 1364, 1415
Dyrenfurth, I., 800, 1773

Eades, C. H., Jr., 801, 1278
Eadie, G. S., 167, 334

Eagle, H., 1519, 1040; 1909, 1968
Earl, A. E., 1522, 1658

Earle, D. P., 124, 217

Easterday, O. D., 65, 106; 500, 111a
Ebaugh, F. G., 1165, 1328
Ebaugh, F. G., Jr., 505, 639

Ebin, L. M., 235, 757

Eble, J. N., 168, 472

Eckfeld, D. K., 1365, 1468

Eckhardt, E., 1551, 1611

Eckhaus, E., 292, 763

Eckstein, H. C., 178S; 742, 1809

Eckstein, J. W., 2032, 681

Eckstein, R. W., 169, 385

Edelhoch, H., 170, 1105

Edelman, I. S., 171, 708

Edelmann, A., 53, 314; 82, 739; 485, 665

Ederstrom, H. E., 172, 630

Edmonds, E. J., 702, 863

Edmonds, L. C., 1659, 1769

Edsall, G., 1916, 2035; 1965, 2070; 2004,
2036

Edsall, J. T., 931, 971

Edwards, C. H., 1785, 1825

Edwards, L. E., 173, 703

Egan, R., 866, 1110

Egerton, J. R., 1350, 1546

Eggman, L., 1220, 1102

Ehrenstein, M. R., 802, 1358

Ehrich, W. E., 1668, 1759

Eich, S., 1026, 1142

Eichel, H. J., 174, 749

Eichelberger, L., 803, 555

Eik-Nes, K. B., 804, 360

Eilberg, R., 1029, 119

Eiler, J. J., 805, 1151

Einbinder, J. M., 175, 317

Einset, B. M., 1829, 1917

Eisenberg, F., Jr., 806, 953

Eisenbrandt, L. L., 33, 732

Elder, T. D., 1644, 1715

Eldred, E., 176, 750

Eldrup-Jgrgensen, Sv., 177, 751

Eliasson, 8S. G., 606, 244

Elion, G. B., 807, 936

Elisberg, B. L., 1935, 2034

Ellenbogen, E., 1188, 1103

Ellenbogen, L., 808, 1785

Ellinger, F., 1366, 26

Elliott, H. W., 1281, 1497

Elliott, W. H., 1000, 851

Ellis, C. H., 178, 752

Ellis, J. P., 21, 512

Ellis, J. P., Jr., 179, 681; 643, 680

Ellis, M. E., 1340, 71

Ellman, G. L., 1367, 1559

Elmadjian, F., 180, 649

Elrick, H., 1182, 1153

Elvehjem, C. A., 992S; 1867S; 1825, 1858

Elwyn, D., 928, 1317

Ely, T. 8., 181, 719

Embree, N. D., 1809, 1847

Emerson, G. A., 1636, 1549 :

Emerson, Gladys A., 1709, 67; 1786, 1889

Emerson, G. M., 182, 265

Emerson, J. D., 182, 265

Enders, A. C., 83, 414

Engel, F. L., 183, 195

Engel, L. L., 966, 1268; 1134, 1160

Engel, R. W., 1262, 1893

England, R. W., 5, 510

Englard, S., 809, 1149

Engle, R. R., 1135, 820

Englesberg, E., 1910, 1208

Enns, T., 755, 215

Enroth, C., 204, 527

Entenman, C., 184, 659; 249, 77

Epanchin, V., 200, 309

Erdés, E. G., 1289, 172; 1368, 1639; 1568,
174

Erlich, F., 89, 371

Ernster, L., 726, 1013

Ershoff, B. H., 1783, 1838
Ervin, F. R., 84, 328
Eschenbrenner, A. B., 1669, 1719
Esser, B., 1786, 1889

Essex, H. E., 602, 519

Essig, A., 318, 459

Essig, C. F., 185, 379; 1377, 1505
Estabrook, R., 810, 1235

Evans, C., 1323, 954

Evans, E. A., Jr., 872S; 855, 22
Evans, F. G., 186, 424

Evans, H. M., 1787, 1817; 1844, 1827
Evans, J. D., 187, 660

Evans, R. A., 1357, 1533
Evarts, E. V., 188, 534

Everett, G. M., 1369, 1427; 1599, 1575
Everett, J. W., 189, 413
Eversole, W. J., 190, 348

Ewell, L. H., 1694, 130

Ewy, H. G., 327, 654

Fabing, H. D., 1370, 1672
Fairbairn, D., 1059, 909
Fairbanks, R., 811, 1165
Fairhurst, A. 8., 812, 1364
Fairley, J. L., 894, 1115
Falzone, J. A., Jr., 448, 229
Farah, A., 1371, 1584
Farber, M. B., 1929, 2008
Farr, R. S., 1911, 2002
Farrall, W. R., 295, 336
Farrand, E. A., 575, 677
Farrell, G. L., 191, 362
Featherstone, R. M., 1322, 171; 1388,
1659; 1520, 1471
Fedor, E. J., 1670, 1710
Feeney, G. C., 1372, 1568
Feeney, R. E., 813, 940
Feigen, G. A., 192, 576
Feinberg, H., 342, 769
Feinberg, R., 1912, 2000
Feinstein, R. N., 814, 828
Feist, E., 193, 753
Feldman, H. A., 1913, 2071
Feldman, I., 1359, 1573
Feldman, J. D., 1671, 1729
Feller, D. D., 193, 753
Felts, J. M., 194, 1292
Feng, Y. 8S. L., 1241, 1319
Fenn, W. O., 227, 756a; 231, 756b
Fenton, P. F., 1788, 1901
Ferguson, F. P., 195, 515
Ferguson, I. D., 196, 725
Ferguson, J.,1197, 754
Ferguson, J. K. W., 1364, 1415
Ferguson, R. W., 198, 273
Ferguson, T. M., 1789, 1872
Feringa, E. R., 656, 420
Fernandez-Guardiola, A., 293, 533
Ferris, B. G., Jr., 199, 225
Ferruggia, T., 1343, 1524
Fiala, A., 1672, 1728
Fiala, S., 1672, 1728
Field, J. B., 806, 953; 815, 1054; 816, 100
Figge, F. H. J., 683, 125
Figurovsky, N., 1406, 1538
Filler, D. A., 816, 100
Fillios, L. C., 1763, 8; 1790, 10
Finamore, F. J., 508, 785
Fine, M., 306, 282
Finerty, J. C., 458, 237

FEDERATION PROCEEDINGS

Fink, B. R., 200, 309
Fink, G. I., 843, 1205
Fink, K., 817, 1113; 818, 1114
Fink, R. M., 817, 1113; 818, 1114
Finlayson, J. S., 1791, 1801
Finney, C. R., 160, 333
Fischer, E. H., 956, 1092
Fischer, G. A., 618, 903
Fisher, B., 1670, 1710
Fisher, E. R., 1670, 1710
Fisher, L., 421, 44
Fishman, A. P., 214, 568; 615, 355
Fishman, J. B., 358, 154
Fishman, W. H., 819, 1027
Fiskin, R., 1128, 1126
Fitzgerald, J. E., 820, 112
Fitzhugh, O. G., 1315, 1404; 1373, 1405
Flaks, J. G., 1240, 912
Flavin, M., 821, 1086
Fleisher, J. H., 822, 169
Fleming, D., 401S
Fleming, D. G., 201, 530
Flesher, A. M., 1410, 1607
Flock, E. V., 823, 1321
Florini, J. R., 1227, 832
Flynn, F. V., 752, 1210
Foa, P. P., 202, 711; 203, 755
Fodor, P. J., 824, 56
Fogg, D. E., 1350, 1546
Fogh, J., 825, 1956
Folch, J., 826, 996
Foldes, F. F., 1289, 172; 1353, 173; 1368,
1639; 1374, 176; 1535, 1531; 1568, 174
Foley, J., 1152, 1064
Foley, J. O., 182, 265
Folk, G. E., Jr., 492, 545
Folkers, K., 1852, 1890
Folse, R., 333, 386; 453, 483
Foltz, E. L., 1375, 1553
Forbes, A., 204, 527
Forbes, I., 19; 1292, 1664
Forbes, R. M., 1792, 1902
Formusa, K., 708, 1347
Forster, R. E., 93, 356
Foster, J. W., 1008, 1371
Foster, W. C., 205, 460
Foulkes, E. C., 827, 444
Foulks, J., 206, 298
Fouts, J. R., 1376, 1393
Fowler, D. I., 1871, 1861
Fowler, W. 8., 141, 306
Fowlks, W. L., 1200, 1159
Fox, C. L., Jr., 175, 317
Fox, H., 1874, 1908
Fox, H. M., 1793, 1845
Fox, I. J., 207, 570
Fox, J. J., 828, 840
Fox, J. P., 1914, 2033
Fox, M. R. S., 1767, 1850
Fox, 8. W., 1214, 892
Fox, W., 786, 968
Fradkin, R., 1578, 1446
Fraenkel, G., 835, 1275; 1816, 1276
Francis, R. D., 1669, 1719
Francis, T., Jr., 1903, 1955
Frank, M. H., 208, 564
Frank, S., 135, 643
Franke, F. E., 209, 335
Frankel, S., 1752, 1746
Franklin, E. C., 1237, 976
Franklin, M., 1961, 2073
Frantz, I. D., Jr., 829, 846
Fraser, H. F., 1377, 1505; 1378, 1673
Fraser, I. M., 1379, 1545

Volume 16

Frawley, J. P., 1380, 1403

Fredericks, J., 183, 195

Fredrickson, D. S., 830, 850

Free, A. H., 678, 1264

Free, H. M., 678, 1264

Freeburg, B., 770, 1177

Freedland, R. A., 873, 843

Freeman, G., 242, 186; 331, 307

Freeman, L., 831, 145

Freeman, M. V., 1381, 1394

Freeman, 8., 654, 498

Fregly, M. J., 210, 559; 316, 476

Frenkel, J. K., 1673, 1714

Freter, G. L., 2012, 1977

Freund, J., 1946

Fridovich, I., 832, 1010

Friedemann, T. E., 184S

Frieden, E., 833, 646

Frieden, E. H., 834, 1075

Friedland, B., 1285, 1663; 1510, 63

Friedman, H., 1971, 917

Friedman, J. J., 211, 311

Friedman, M., 213, 756; 1756, 2018; 1915,
1940

Friedman, M. H. F., 212, 611

Friedman, N. B., 1674, 1752; 1748, 1781

Friedman, 8., 835, 1275

Friess, 8S. L., 1604, 187

Frisch, H., 1128, 1126

Fritts, H. W., Jr., 214, 568

Fritz, I. B., 215, 448

Fritz, J. C., 1794, 1839

Froeb, H. F., 216, 684

Froesch, E. R., 217, 588

Froese, G., 218, 634

Frohman, C. E., 1795, 1831

Fromm, H. J., 1382, 1089

Frost, D. V., 1178, 1888; 1855, 1855

Frumin, A. M., 1118, 19°4

Frumin, M. J., 1563, 1430

Fry, R., 1818, 1897

Fryer, J. H., 1796, 1803

Fuentes, J., 1383, 1456; 1462, 1455

Fugazza, J., 1497, 1687

Fujikawa, B., 1379, 1545

Fujimoto, J. M., 1384, 1445

Fukushima, D. K., 836, 1359

Fulchiero, E., 44, 733

Fuld, M., 837, 1029

Fuller, G. R., 219, 596

Fuller, R. K., 1339, 107

Fulton, G. P., 51, M.P., p. 628; 52, 735

Funckes, A. J., 1212, 1345

Funk, C., 824, 56; 838, 1365

Furchgott, R. F., 1385, 1457

Furman, R. H., 839, 66

Furth, J., 1662, 127; 1742, 1748

Futterman, S., 840, 1334

Fuwa, H., 841, 1093

Fuyat, H. N., 1380, 1403

Gabourel, J. D., 1386, 1439
Gabrio, B. W., 1204, 1079
Gaebler, O. H., 842, 1240
Gaensler, E. A., 414, 228
Gaertner, R., 1399, 1705

Gaines, D. 8., 941, 837

Gaines, 8., 1916, 2035; 2004, 2036 _
Gajdusek, D. C., 1969, 1975; 1996, 1992
Gal, E. M., 812, 1364

Galambos, R., 627, 597
Galansino, G., 202, 711; 203, 755
Galdston, M., 220, 357

lume 16

18; 1915,

748, 1781

; 52, 735

March 1956

Galkin, T. W., 387, 324; 388, 773
Gallagher, T. F., 727, 1161; 836, 1359;
1114, 848

Gallo, D. G., 767, 1775

Galum, A. B., 835, 1275

Ganis, F. M., 1675, 1166

Gaon, J., 2014, 1989

Garcia-Arocha, H., 264, 146
Gardella, J. W., 221, 79

Gardier, R. W., 1387, 1523
Gardiner, R. C., 1165, 1328

Garner, R. L., 843, 1205

Garren, H. W., 222, 313; 1811, 15
Garst, J. B., 844, 406

Garvey, J. S., 1917, 2025

Garvin, J. E., 1797, 1006

Gaskins, J. R., 1530, 1627

Gass, G. H., 1222, 1649

Gatt, 8., 845, 1008

Gauchat, R. D., 1388, 1659

Gaunt, R., 106, 516

Geddes, I. C., 846, 1477

Geer, J. C., 1676, 1744

Gehenio, P. M., 223, 625

Geiger, A., 224, 375

Geiger, G. L., 1921, 1794

Geiger, R. S., 225, 378

Geiling, E. M. K., 1295, 1591
Geller, D. M., 847, 1018

Geller, J., 220, 357

Gemmill, C. L., 1389, 463; 1390, 1674
Genest, KX., 1445, 175

Gennaro, J. F., Jr., 226, 390
Genuth, S. M., 785, 875

George, R., 1281, 1497; 1391, 1588
Gerard, R., 279S

Gerber, P., 1928, 2011

Gergely, J., 848, 1104

Gerhardt, P., 1914, 2033

Gerheim, E. B., 1918, 2058
Gerschman, R., 227, 756a; 231, 756b
Gershberg, H., 228, 522

Gershoff, S. N., 1798, 1926
Gershon-Cohen, J., 1014, 1372; 1836, 1915
Gerst, P. H., 469, 429

Gerstner, H. B., 76, 134; 229, 105
Gesler, R. M., 1392, 1525

Gest, H., 1064, 958

Gey, G. O., 455, 398

Gey, M. K., 455, 398

Ghosh, J. J., 849, 1169

Giarman, N. J., 1393, 1632

Gibbs, M., 1096, 906

Gibson, D. M., 850, 1287

Giebisch, G., 280, 219

Gifford, G. E., 1919, 1969

Gilbert, D. L., 227, 756a; 231, 756b
Gilbertson, E. V., 914, 1136

Gill, T. J. IIL, 566, 619; 1677, 1788
Gilmore, J. P., 232, 670

Gilmore, L. O., 205, 460

Gilmour, C. M., 1238, 1379
Gilvarg, C., 851, 1304

Gimbel, N. S., 1851, 1856

Ginoza, W., 1120, 1044

Ginsberg, H. S., 1920, 1942; 2025, 2069

Ginsburg, V., 852, 925

Ginski, J. M., 233, 618

Girerd, R. J., 234, 310

Gitlin, D., 853, 84

Glaser, G. H., 580, 471

Glass, G. B. J., 235, 757; 236, 609; 1589,
1641

Glassman, J. M., 1394, 1485

AUTHOR INDEX

Glaubach, 8., 1641, 137
Glazko, A. J., 155, 746; 237, 1478
Glenn, J. L., 238, 758; 854, 1231
Glinos, A. D., 239, 455
Glitzer, M. S., 240, 464
Goebel, W. F., 1556
Gofman, J. W., 1291, 88
Gogerty, J. H., 1395, 1651
Goh, K., 241, 204
Golbey, M., 1396, 1622
Gold, A. J., 242, 186
Gold, G. L., 566, 619
Gold, H., 1404, 1555
Goldbaum, L. R., 1397, 1442
Goldberg, H., 243, 285
Goldberg, L. I., 1398, 1416
Goldensohn, E., 1563, 1430
Goldfeder, A., 244, 33
Goldfine, H., 855, 22
Goldhamer, R. E., 619, 657
Goldie, H., 1921, 1794; 1922, 131
Goldin, A., 554, 1212
Goldman, A., 1328, 1482
Goldman, D. E., 181, 719
Goldman, D. 8., 856, 1219
Goldman, P., 245, 438
Goldrich, A. D., 1882, 1876
Goldsmith, E. D., 246, 759; 1223, 1920;
1799, 1814
Goldstein, L., 1497, 1687
Goldstein, M. H. Jr., 353, 600
Goldstein, M. 8., 55, 458; 247, 361
Goldsworthy, P. D., 248, 807
Goldthwait, D. A., 857, 911
Goldwasser, E., 858, 1253
Goldwater, W. H., 249, 77
Gollan, F., 250, 338
Gollub, S., 251, 45
Golubow, J., 859, 1034
Gong, J. K., 1339, 107
Gongaware, M. 8., 204, 527
Gonzalas, L., 1883, 1916
Gonzalez, I. E., 839, 66
Gomoll, A. W., 1453, 1487
Goodall, McC., 356, 655
Goodgal, S. H., 1923, 2059
Goodkind, M. J., 22, 214; 252, 280
Goodland, Ruth, L., 1645, 1758
Goodman, H., 1399, 1705; 1908, 2046
Goodman, H. C., 1300, 1755
Goodman, I., 860, 841
Goodman, M., 1924, 1981
Gordon, A. 8S., 53, 314; 485, 665; 601, 793
Gordon, H. H., 1846, 1874
Gordon, J., 542, 974
Gordon, J. G., 1921, 1794
Gordon, S. M., 1309, 1593
Gosselin, R. E., 253, 667; 1386, 1439
Gotch, F., 171, 708
Gotterer, G., 1258, 897
Gottesman, L., 772, 1172
Goucher, C. R., 861, 1366
Gould, R. G., 862, 1367
Gourley, D. R. H., 254, 399
Gourzis, J. T., 1348, 1633
Govier, W. M., 1551, 1611
Grabar, P., 1181, 1958
Grabowski, R., 1254, 1065
Grace, J. B., 207, 570
Grace, R. A., 563, 672
Grad, B., 17, 323
Graeme, M. L., 1400, 1418
Graf, L., 1093, 1960

vii

Graham, J. B., 1951, 160

Graham, L., 208, 564

Gran, F. C., 783, 1280

Grande, F., 255, 500; 428, 505a; 603, 505b

Grannis, G. F., 1145, 973

Grant, W. C., 392, 624

Gravenstein, J. S., 1401, 1496

Graves, J. L., 978, 818

Gray, B. L., 1324, 1388

Gray, C. L., 719, 658

Gray, E. H., 1612, 1612

Gray, I., 863, 1308; 1397, 1442

Gray, W. D., 1402, 1540

Grayston, J. T., 1756, 2018

Green, A. A., 1017, 960

Green, D. E., 974, 1016

Green, D. M., 234, 310; 1557, 83; 1558, 1695

Green, F. O., 814, 828

Green, H. D., 142, 652; 145, 745; 1472, 1425

Green, I., 256, 216

Green, J. D., 258, 416

Green, J. W., 257, 621

Green, M., 864, 1117

Green, 8., 819, 1027

Green, V. A., 1403, 1675

Greenberg, D. M., 926, 1020; 1085, 883

Greenberg, L. D., 865, 1832; 1832, 1934

Greene, C. 8., 286, 761

Greene, L. C., 259, 724; 378, 432

Greenstein, J. P., 718, 1808

Greenwald, I., 1800, 1929

Greep, R. O., 359, 520

Gregg, N. C., 1925, 1941

Gregory, J. D., 847, 1018

Greif, R. L., 260, 347

Greiner, T., 1404, 1555

Grenan, M. M., 562, 161

Grenell, R. G., 261, 320

Griesemer, E. C., 1405, 1489

Griffin, O. R., 1735, 1

Griffith, W. H., 179S

Grimmett, P., 698, 1076

Grindlay, J. H., 823, 1821

Grisolia, S., 1217, 1215

Gross, E. G., 1635, 1598

Gross, E. M., 866, 1110

Gross, J., 240, 464; 262, 1200

Gross, P., 1678, 1768

Gross, P. R., 263, 640

Grossberg, A. L., 264, 146

Grossman, A. J., 1298, 1621; 1397, 1622;
1406, 1538

Grossman, L., 867, 817

Grossman, R. G., 265, 271

Grubbs, R. C., 266, 101

Gruber, C. M., Jr., 1833, 1464; 1407, 1465

Grumbach, L., 267, 490

Grundfest, H., 335, 248; 476, 377

Gubler, C. J., 1801, 1887

Guccione, I., 672, 802

Guest, M. M., 451, 208; 948, 118

Guillebeau, J., 1729, 1738

Guillemin, R., 268, 366

Guilmain, J., 1691, 1899; 1730, 1800

Gundersen, K., 269, 318

Guroff, G., 1133, 1139

Gutensohn, M. O., 149, 553

Gutman, A. B., 36, 218; 270, 349; 1247,
919

Gutmann, H. R., 868, 94

Guyton, A. C., 271, 425

Guzman-Flores, C., 293, 533

Gyenes, L., 542, 974

Gyorgy, P., 1112, 1204

vill

Habel, K., 1909, 1968; 1925, 1941

Habermann, R. T., 1731, 1764

Habif, D. V., 547, 700

Haddy, F. J., 272, 426

Hafez, E. 8. E., 667, 411

Haff, R. F., 1926, 1938

Hafkenschiel, J. H., 533, 383

Haft, D. E., 1679, 1059

Hageman, E., 873, 843

Hagen, P., 1408, 170

Hagerman, D. D., 1228, 1290

Hahn, J. W., 273, 81

Haist, R. E., 274, 36

Halberg, F., 107, 644

Hale, C. J., 340, 550

Hale, D. B., 1699, 73

Haley, K., 1643, 1745

Haley, T. J., 1358, 1615; 1409, 1676; 1410,
1607

Hall, F. G., 314, 230

Hall, L. M., 869, 1298

Hall, W. K., 1802, 1842

Hallesy, D. W., 1411, 1677

Halliday, 8. L., 1218, 98

Halpern, E., 1707, 1777

Halsey, Y. D., 1738, 1154

Halsted, J. A., 1875, 1841

Halver, J. E., 1782, 1864; 1803, 1898;
1804, 1930

Hamilton, H. B., 1036, 1228

Hamilton, J. R., 274, 536

Hamilton, L. H., 308, 764

Hamilton, M. G., 1066, 1042

Hammel, H. T., 275, 632

Hammon, W. M., 1927, 1993; 1933, 1991;
1957, 1976

Hampton, J. K., Jr., 276, 23

Hamre, D., 1928, 2011

Hamwi, G. J., 1649, 24

Hanahan, D. J., 870, 1001

Handler, A. H., 1680, 1747

Handler, P., 832, 1010

Handley, C., 1450, 1586

Handley, C. A., 1591, 1585

Handschumacher, R. E., 871, 96

Hane, D. L., 1304, 1625

Hankes, L. V., 872, 1175; 890, 1174

Hanna, C., 1412, 1606

Hanna, L., 1937, 2017

Hansbury, E., 989, 1072

Hansen, A. E., 1884, 1852

Hansen, R. G., 873, 843

Hansl, N. R., 874, 824

Harary, I., 875, 858

Harbers,E., 884, 52

Hardin, C. A:, 1750, 1771

Hardinge, M. G., 1413, 1483

Hardy, J. D., 289, 722; 587, 723

Hare, A., 1870, 1903

Hare, K., 277, 590

Hare, R. 8., 277, 590

Harkness, W. D., 1414, 1605

Harman, J. W., 1681, 1795

Haroutunian, L. M., 333, 386

Harper, L., 1805, 1912

Harper, P. V., 278, 43

Harrington, H., 876, 51

Harris, A. 8., 279, 760; 280, 494

Harris, J., 877, 931

Harris, P. L., 1809, 1847

Harris, P. N., 1545, 1396

Harris, R. 8., 1880, 1925

Harris, 8., 2030S; 1929, 2008

Harris, T. N., 2030S; 1929, 2008

FEDERATION PROCEEDINGS

Harrison, F. M., 407, 688

Harrow, B., 991S

Harshman, S., 1135, 820

Hart, E. R., 281, 376; 1467, 1574

Hart, H. E., 1116, 1129

Hartline, H. K., 482, 528

Hartman, F. W., 42, 575; 1682, 1720

Hartman, 8. C., 878, 910

Hartmann, R. C., 553, 152

Hartnett, C., 775, 1357

Hartroft, W. 8., 1683, 1774

Harvey, J. C., 282, 554

Harvey, R. B., 284, 481

Harvey, R. F., 283, 352

Hash, J. H., 1806, 1095

Hass, G., 114, 272; 1640, 1702; 1652, 1707;
1741, 68

Hassid, W. Z., 852, 925

Hasselmeyer, E., 1871, 1861

Hastings, A. B., 279S; 691, 1063

Hatefi, Y., 905, 1242

Hauck, H. M., 1807, 1914

Haugaard, N., 296, 1473; 879, 904

Hauge, J. G., 706, 1214

Haugen, M. G., 295, 336

Haurowitz, F., 880, 810; 1980, 2067

Hausberger, B. C., 285, 200

Hausberger, F. X., 285, 200

Hauser, G., 928, 1317

Haverback, B. J., 1415, 1638

Hawk, E. A., 1887, 1892

Hawkins, J. R., 1370, 1672

Hawley, E. E., 1808, 1931

Hawthorne, E. W., 286, 761; 307, 562;
659, 60

Hayaishi, O., 929, 1084

Hayano, M., 723, 1158

Hayden, R., 78, 685

Hayes, H. B., 321, 699

Hayes, W. J., Jr., 1363, 1407

Haynes, F. W., 371, 207

Haynes, R., C., Jr., 881, 1078

Hays, H. W., 1286, 1582; 1416, 1609

Haywood, C., 400S

Hazelton, L. W., 1612, 1612

Hearn, W. R., 268, 366

Heaton, R. W., 1394, 1485

Hecht, L. I., 882, 1184

Hechter, O., 380, 315; 588, 1291

Heck, W. W., 883, 1302

Hegsted, D. M., 1677, 1788; 1798, 1926;
1879, 1904; 1883, 1916

Heidelberger, C., 884, 52

Heidelberger, M., 885, 1957

Heilbrunn, L. V., 276S

Heimer, R., 886, 1202

Heinrich, M., 1417, 1402

Heinrich, R., 287, 123

Heinz, R., 887, 614

Heiselt, L. R., 1200, 1159

Heisey, 8. R., 1418, 1399; 1559, 1400

Heisler, C. R., 888, 1329

Heisse, C. K., 1623, 1624

Heller, J. H., 288, 666

Hellman, L., 727, 1161; 1114, 848

Helmer, O. M., 889, 558

Heming, A. E., 1337, 1536; 1430, 1578;
1479, 1534

Hemingway, A., 557, 358

Henderson, L. M., 872, 1175; 890, 1174

Henderson, R. B., 817, 1113

Hendler, E., 289, 722

Hendley, C. D., 1305, MP, p. 628; 1419,
1600

—

Volume if

Hendrickson, M. J., 1441, MP, p. 628
Henley, M. R., 844, 406

Henley, R. M., 1794, 1839
Henneman, D. H., 1421, 1515
Hennessy, A. V., 1907, 2014; 1930, 2013
Hennigar, G. R., 1457, 1470
Henry, J. P., 290, 688

Henschke, U. K., 1723, 166
Heppel, L. A., 891, 1256

Herbst, E. J., 1242, 867

Herezeg, 8. A., 1549, 1527
Hernandez, T., 291, 762; 769, 487
Hernandez-Peon, R., 292, 763; 293, 533
Herndon, J. F., 1813, 1829
Heroux, O., 294, 541

Herr, E. B., Jr., 892, 828
Herranen, A. M., 893, 1269
Herrick, J. F., 295, 336

Herring, A. 8., 2001, 1990
Herriott, R. M., 1923, 2059
Herrmann, R. L., 894, 1115
Herschberger, R., 1455, 1603
Hershberger, L. G., 1422, 1620
Hershey, 8. G., 672, 802; 1554, 1693
Herting, D. C., 1809, 1847
Hertzman, A. B., 125, 650; 196, 725
Herzog, E., 1716, 1772

Hess, M. E., 296, 1473

Hess, S. M., 1423, 1631

Hester, C. L., 1659, 1769

Hetzel, P. 8., 352, 569

Hiatt, E. P., 297, 346

Hickey, M. D., 717, 1844

Hicks, M. L., 1407, 1465

Hien, L. T., 1424, 1480

Hiestand, W. A., 446, 778

Hift, H., 1196, 989

jac Ma= Mes MesBasMas Macias [i a-Maof«.ooMa:Ba-Ma-I«-1-.B<cE«>Ih«-I«>ih<sih<-I«-I«-I--9h-. 0h --0h--0h--0- <9 -- 0k --0h-- In --- -]

Higginbotham, R. D., 298, 148 H
Higgins, E. S., 895, 1883 H
Higgins, J. A., 116, 606 H
High, E. G., 1810, 1885 H

Highman, B., 7, 511; 1684, 1716; 1m,) 2

1717 H
Hilgar, A. G., 1710, 1798 H
Hill, C. H., 1811, 15 H
Hill, H. E., 1377, 1505 H
Hill, M. S., 589, 182 H
Hill, R. G., 1297, 121; 1425, 150 H
Hill, R. J., 896, 902; 947, 1818 st
Hills, C. H., 1073, 922 it
Hillyard, I. W., 1426, 1459 Hi
Hilton, J. G., 1427, 1608 Hi
Hilton, J. L., 897, 1368 i
Himwich, H. E., 647, 269 i
Himwich, W. A., 299, 374 Hi
Hinds, H., 1106, 1208 Hi
Hine, C. H., 1521, 1689 -

Hines, H. M., 593, 1032
Hiraki, G. Y., 1092, 447 Hi

Hiratzka, T., 1366, 1879 Hi
Hirs, C. H. W., 8714S it
Hirschmann, H., 766, 1354 i
Hitchings, G. H., 807, 936 :
Hobart, J., 1115, 1877 “

Hobby, G., 739, 933
Hoch, F. L., 300, 885; 2619, 1099 Hi

Hoch-Ligeti, C., 1685, 1737 Hi
Hock, R. J., 301, 544 i
Hoffman, B. F., 302, 487 Hi
Hofmann, K., 1003, 1285 Ht
Hogan, A. G., 1847, 1822 Mh
Hogans, A. F., 1625, 1088 Hi
Hogben, C. A. M., 129, 443; 1561, 1668 -

Hogg, J. F., 898, 943

7olume tj

P, p. 628

1930, 2013

3; 293, 583

0
1, 1693

6, 725

1716; 1721,

61, 1643

March 1956

Hokama, Y.;-1543, 128

Hokin, L. E., 899, 1472

Holaday, D. A., 303, 686

Holburn, R. R., 98, 153

Holden, J. T., 900, 860

Holland, W. C., 1287, 1562; 1428, 1412

Hollander, F., 304, 615

Hollett, C., 305, 89

Holley, H., 1070, 1770

Hollinger, G. W., 609, 525

Hollinshead, A. C., 1429, 1552

Hollis, V., Jr., 220, 357

Hollmann, 8., 1216, 945

Holloway, R. J., 1317, 1650

Holman, R. L., 1676, 1744; 1735, 1

Holman, R. T., 1065, 1853

Holt, J. P., 306, 282

Holt, L. E., Jr., 1871, 1861

Holtkamp, D. E., 1337, 1536; 1430, 1578;
1479, 1534

Hong, 8. 8., 402, 702

Hook, A. E., 1993, 1953; 2016, 1951

Hoover, C. R., 901, 1791

Hope, J. M., 180, 649

Hopkins, E. L., 307, 562

Hoppe, J. O., 1392, 1525

Hopps, H. E., 1899, 1988

Horecker, B. L., 902, 947

Horowitz, H. B., 318, 459

Horowitz, N. H., 870 S

Horvath, S. M., 308. 764; 575, 677

Horwitt, M. K., 1812, 1813

Horwitz, B. S., 1431, 1570

Hosko, M. J., 1432, 1656

Hosoya, E., 1433, 1678

Houck, C. R., 157, 747; 309, 563

Houde, R. W., 1434, 1501; 1611, 1506

Housholder, D. E., 268, 366

Hove, E. L., 1813, 1829

Howard, E., 310, 316

Howard, J. D., 465, 302

Howe, C., 1931, 2043; 1972, 2016

Howe, J., 1777, 1862

Howell, G. L., 625, 796

Howes, D., 1985, 1970

Howland, J. W., 420, 138

Howton, D. R., 1023, 1293

Hoyt, R. E., 1674, 1752

Hrenoff, A. K., 1435, 1679

Hsia, 8. L., 903, 852

Hsiung, G. D., 1932, 1972

Hsu, J. M., 1814, 1826

Huang, K. C., 1436, 1481

Hubbard, R., 904, 961

Huber, R. deV. 1302, 1657

Huckabee, W. E., 311, 579

Huebscher, R. G., 45, 635

Huennekens, F. M., 905, 1242; 1204, 1079

Huf, E. G., 312, 391

Huff, R. L., 193, 753

Huggins, R. A., 361, 571

Hughes, J. R., 188, 534; 313, 532; 387, 324;
388, 773

Hughes, L. B., 1699, 73

Hulcher, F. H., 1815, 908

Hull, W. E., 314, 230

Hundley, J. M., 1024, 1055

Hunter, D. H., 1893, 2052

Hunter, F. E., Jr., 906, 986

Hunter, F. R., 508, 785

Hunter, J., 739, 933

Huntsman, D. B., 1430, 1578

Hurd, M. 8., 192, 576

Hurlbut, W. P., 315, 396

AUTHOR INDEX

Hurley, L. A., 1686, 1704
Hurwitz, J., 907, 946
Hurwitz, L., 1414, 1605
Hussey, C. V., 908, 35
Hutcheon, D. E., 1564, 1484
Hutcheson, R. M., 1216, 945
Hutchings, B. L., 1169, 1247
Hutchison, D. J., 695, 1263; 909, 1262
Hutt, B. K., 308, 764
Hwang, Kao, 1437, 1640
Hyman, C., 457, 410

| ror P. F., 88, 506; 316, 476
Ibsen, K., 910, 21
Thrig, J., 1578, 1446
Iman, I. Z. E., 195 1991
Imboden, J., 1468, 1466
Inamine, E., 1191, 1307
Ingersoll, F., 686, 1271
Ingold, A. H., 539, 264
Inscoe, J. K., 1478, 1684
Intoccia, A., 317, 478
Iriye, T. T., 838, 1365
Irvine, E. 8., 1832, 1934
Irvine, J. E., 1685, 1787
Irvine, K., 1685, 1737
Irvine, W., 735, 1193
Irwin, 8., 1438, 1653
Isaacs, J. P., 1401, 1496
Isaacs, M. C., 318, 459
Isbell, H., 1377, 1505; 1439, 1680; 1400,
1661
Ishida, N., 1934, 2009
Ishii, K., 911, 915
Isselbacher, K. J., 684, 949
Ito, S., 1687, 1796
Ito, T., 1816, 1276
Itoh, S., 319, 440; 320, 765
Itskovitz, H., 879, 904
Ivy, A. C., 321, 699; 389, 2
Ivy, E. K., 321, 699
Izzo, A. J., 912, 1350

Heitinsis D. P., 322, 155
Jackson, E. B., 1899, 1988; 1935, 2034
Jacob, M. I., 850, 1287
Jacobs, E. E., 913, 1011
Jacobs, F. A., 914, 1136
Jacobs, G. 8., 260, 347
Jacobson, K. B., 915, 988
Jaenicke, L., 916, 1330
Jaffé, E. R., 992, 622
Jahnke, J. K., 1817, 1863
Jahrmarker, H., 885, 1957
Jailer, J. W., 323, 405
Jakoby, W. B., 917, 1223
James, T. W., 381, 1047
Jameson, E., 324, 126
Jandorf, B. J., 1253, 819
Jang, R., 987, 924

Jaques, D. A., 325, 561
Jaques, L. B., 421, 44
Jaques, W. E., 1688, 1779
Jardetzky, C. D., 918, 982
Jaroslow, B. N., 1936, 159
Jason, R. 8., 307, 562
Jasper, R. L., 147, 450; 383, 543
Jawetz, E., 14225; 1937, 2017
Jeanloz, R. W., 919, 1310
Jeffay, H., 920, 809

Jeffries, H., 2024, 1996

Jelinek, B., 1119, 811

Jenden, D. J., 1604, 187

Jenkins, W. T., 921, 1030

Jennings, R. B., 1689, 1740

Jensen, H., 922, 41

Jensen, K. E., 1938, 2012

Jensen, L. S., 1818, 1897

Joardar, 8. N. D., 326, 729

Jochim, K. E., 327, 654

Jodrey, L. H., 1441,MP, p. 628

Johnson, A. R., 328, 470

Johnson, B. C., 177S; 965, 1279; 1775, 1811

Johnson, C. A., 777, 1363

Johnson, D. H., 1487, 1547

Johnson, H. D., 329, 766

Johnson, J. A., 330, 641

Johnson, L. H., 1058, 1062

Johnson, R. B., 923, 1189

Johnson, R. E., 1691, 1899; 1692, 1797;
1730, 1800

Johnson, R. P., 115, 232; 331, 307

Johnson, S. A., 1151, 40

Johnson, W. J., 924, 97

Johnston, C. G., 332, 662

Johnston, F. A., 1819, 1922

Johnston, J. M., 863, 1308

Jolly, E. R., 1442, 1431

Jones, D. 8., 463, 209

Jones, F. T., 724, 1274

Jones, J. E., 518, 1356

Jones, M., 1322, 171

Jones, M. E., 773, 1299

Jones, R. L., 925, 1195

Jones, R. S., 1656, 1726; 1690, 1725

Joralemon, J., 656, 420

Jordan, D. L., 628, 28

Jordan, W. S., Jr., 1939, 1939

Jude, J. R., 333, 386; 453, 483

Jukes, T. H., 1820, 1843

Juras, D. 8., 1706, 1781

Katat, E. A., 1931, 2043; 1966, 2042
Kabara, J. J., 1511, 108

Kagawa, C. M., 1443, 1619

Kahn, J. B., Jr., 1444, 1507; 1608, 1411
Kahn, R. L., 1940, 2027

Kalckar, H. M., 684, 949

Kalf, G. F., 1269, 1311

Kalfayan, B., 1941, 1963

Kalow, W., 1445, 175

Kandel, A., 1446, 1589

Kanter, G. S., 334, 721

Kaplan, R., 1647, 1778
Kaplan, 8. A., 339, 687
Kappell, B., 1343, 1524
Kapphahn, J. I., 991, 1211
Karasek, M. A., 926, 1020
Karler, A., 927, 1043

Karler, R., 609, 525
Karnofsky, D. A., 774, 95
Karnovsky, M. L., 928, 1317
Karpovich, P. V., 340, 550
Karush, F., 1960, 975
Karvinen, E., 389,

Katsh, S., 341, 768

Katsuyama, D. M., 1098, 806

Katz, J., 930, 1057; 1997, 449

Katz, L. N., JS, p. 628; 342, 769; 578, 792

Katzman, P. A., 1097, 1272

Kaufman, B. D., 343, 694

Kaunitz, H., 1691, 1899; 1692, 1797; 1730,
1800

Kavaler, F., 344, 573

Kay, C. M., 931, 971

Kay, E. R. M., 345, 981

Kay, J., 1399, 1705

Kay, L., 905, 1242

Kay, W. W., 2013, 1952

Kazenko, A., 155, 746

Kearney, D. B., 932, 1233; 1011, 1234

Kearns, C. W., 984, 966

Keasling, H. H., 1635, 1598

Keats, A. S., 1447, 1502

Keech, B., 1051, 1017

Keith, E. F., 1448, 1513

Keitzer, W. F., 325, 561

Keller, A. D., 346, 524

Keller, D. M., 394, 290

Keller, E. B., 986, 879

Keller, P. J., 933, 895

Kelley, L., 1821, 1913; 1922, 131

Kellogg, R. H., 347, T70

Kelly, A. R., 1682, 1720

Kelly, E. M., 1316, 138a

Kelly, K. H., 1449, 626

Kelly, L. 8., 348, 20

Kelly, M. G., 1530, 1627

Kelso, A. F., 349, 304

Kendall, F. E., 1991, 1782

Kendrick, J. E., 350, 651

Kennedy, E. P., 1246, 1003

Kenney, F. T., 934, 987

Kensler, C. S., 1598, 1544

Kent, B., 1450, 1586; 1591, 1585

Kent, G., 1716, 1772

Kent, J. F., 1943, 1980

Keplinger, M. L., 1451, 1469

Kern, M., 935, 825

Kersting, D. J., 563, 672

Kessler, R. H., 351, 343

Keston, A. S., 936, 963

Kety, 8. S., 565, 372

Keutmann, E. H., 912, 1350

Kewitz, H., 937, 189

Keyl, A. C., 1452, 1596

Keys, A., 255, 500; 428, 505a; 558, 491; 603,
505b; 1762, 3

Keys, J. R., 352, 569

Kiang, N. Y. S., 353, 600

Kien, G. A.7 1453, 1487

Kies, M. W., 938, 1724

Kilbourne, E. D., 1944, 2010

Killam, K. F., 1454, 1652

Kim, 8. H., 1940, 2027

Kimeldorf, D. J., 441, 508; 1299, 29

Kimmel, H. B., 1145, 973

Kimmel, J. R., 939, 1108

Kimmelstiel, R., 1228, 1290

Kimura, 8. J., 1937, 2017

Kinard, F. W., 354, 661

Kinard, S., 1455, 1603

King, C. G., 1122, 955

King, C. T. G., 355, 715

King, J. 8., Jr., 801, 1273; 1456, 1441

King, J. T., 379, 394

King, K. W., 1806, 1095; 1815, 908

King, T. E., 940, 1238; 943, 907

King, W. M., 1577, 1440

FEDERATION PROCEEDINGS

Kinnory, D. 8., 941, 837
Kinosita, R., 1714, 133
Kirshner, J., 1617, 1450
Kirshner, N., 356, 655
Kisliuk, R. L., 942, 1244
Kissel, J. W., 1330, 1432
Kitos, P. A., 943, 907
Kitzinger, C., 45, 635; 425, 636
Kiyasu, J. Y., 357, 446
Klegman, J. H., 20, 730
Klein, G., 458, 237
Klein, J. R., 944, 830
Klein, P. D., $45, 1181
Kleitman, N., 144, 418
Kline, B. E., 182S
Kline, D. L., 358, 154
Klingenberg, M., 751, 1236
Klinger, O. J., 522, 580
Klingman, G. I., 1457, 1470
Kniazuk, M., 1349, 1580
Knight, H. B., 1692, 1797
Knipp, E. A., 1976, 1979
Knobil, E., 359, 520
Knoefel, P. K., 1436, 1481
Knox, W. E., 1071, 964
Kobayashi, T., 644, 384
Koch, A., 1458, 1539; 1631, 1426
Koch, A. L., 946, 1122; 1736, 1254
Kochakian, C. D., 608, 409
Kocholaty, W., 861, 1366
Koesis, J. J., 1295, 1591; 1610, 1438
Kodama, J. K., 1459, 1681
Koeppe, R. E., 896, 902; 947, 1318
Koffler, H., 1984, 1984
Kohler, H., 848, 1104
Kohn, R. R., 1945, 1965
Koizumi, K., 592, 484
Koler, J., 665, 354
Kolff, W. J., 385, 80
Kolmen, 8. N., 948, 118
Koltun, W. L., 820, 112
Kominz, D. R., 360, 260
Konigsberg, W. H., 1193, 1037
Koniuszy, F. R., 1852, 1890
Konnerth, A., 420, 138
Kopeloff, L. M., 111, 741
Kopeloff, N., 111, 741
Koppanyi, T., 1372, 1568; 1516, 1626
Koppel, J. L., 949, 34; 1642, 39
Koppelman, R., 855, 22
Koprowski, H., 1946, 2072a
Korman, 8., 1328, 1482
Korn, E. D., 1062, 1196
Kornberg, A., 950, 1185
Kornetsky, C., 565, 372; 1460, 1662
Korngold, L., 1947, 1982
Korol, B., 1461, 1410
Korzenovsky, M., 951, 855
Koshland, D. E., Jr., 892, 823
Kouwenhoven, W. B., 424, 495
Kozloff, L. M., 952, 1187
Kraintz, L., 361, 571
Kramer, E. R., 1334, 1637
Kramer, 8., 246, 759
Krampitz, L. O., 953, 1227
Krantz, J. C., Jr., 1321, 1599; 1602, 1514;
1623, 1624
Krasna, A. I., 954, 959
Kratz, P., 1543, 128
Kratzer, F. H., 1822, 1859
Kraus, 8S. D., 362, 340
Krause, R. F., 955, 1834
Kraybill, H. F., 184S
Krayer, O., 1462, 1455

Volume 16

Krebs, E. G., 956, 1092
Kremen, D., 397, 321
Kretchmer, N., 363, 453
Kreuzer, F., 364, 353
Krevans, J. R., 322, 155
Krezanoski, J. Z., 805, 1151
Krieger, H., 549, 591
Krieger, H. P., 632, 535
Krimsky, I., 845, 1008
Krinsky, N. I., 365, 1348
Krnjevié, K., 366, 327
Kroe, R. L., 367, 235
Kropa, E. L., 1370, 1672
Krueger, H., 368, 771; 369, 645
Krueger, R. C., 957, 956
Krum, A. A., 609, 525
Kruse, R., 1371, 1584
Kubicek, W. G., 370, 565
Kuby, S. A., 1050, 1026
Kuck, J. F. R., Jr., 332, 662
Kuhn, W. L., 1463, 1520
Kuhns, W. J., 1948, 1961
Kuida, H., 371, 207
Kuluz, M., 1527, 1458
Kulwich, R., 1823, 1881
Kumm, M. G., 149, 553
Kun, E., 958, 1226

Kuna, 8., 1349, 1580
Kundin, W. D., 1464, 1551
Kunkel, A. M., 1626, 188
Kunkel, H. G., 1237, 976
Kunkel, H. O., 959, 547
Kuntzman, R., 1465, 1630
Kupsky, C. H., 2016, 1951
Kuron, G., 1876, 9

Kurtz, G. W., 1772, 1844
Kurtz, L. H., 362, 340
Kusama, T., 176, 750
Kushner, D. 8., 1716, 1772
Kutnerian, K., 960, 1132
Kuyper, A. C., 960, 1132
Kwit, N., 1404, 1555
Kwong, E., 1777, 1862
Kyker, G. C., 961, 109

La Du, B. N., 1277, 1170

Lagerborg, D. L., 1979, 1995

Lahr, T. N., 962, 1320

Laird, A. K., 1693, 979

Lajtha, L. G., 1082, 1182

Laken, B., 480, 299

Lalich, J. J., 1705, 1767

Lambert, E. H., 141, 306

Lambert, G. F., 1855, 1855

Lambertsen, C. J., 123, 300; 343, 694;
1466, 1504; 1619, 1503

La Motte, L. C., 1905, 1994

Lampen, J. O., 963, 939

Lamport, H., 372, 728

Lamson, B. G., 1648, 186; 1694, 130

Lamson, E. T., 180, 649

Lamy, F., 964, 42

Landau, B. R., 373, 546

Landers, R. M., Jr., 621, 393

Landesman, E., 220, 357

Landowne, M., 448, 229

Landy, M., 1916, 2035; 1949, 2037; 2004,
2036

Lane, M. D., 965, 1279

Lane, W. B., 1339, 107

Lang, J., 1092, 447

Lang, S., 1977, 678

ume 16

March 1956

langdon, R. G., 1208, 898
langen, L., 981, 1106
langer, L., 966, 1268
langfitt, T. W., 281, 376; 1467, 1574
langner, R. R., 374, 1135
lanphier, E. H., 375, 696
lansing, A. I., 278S
lapresle, C., 1181, 1958
larks, 8S. D., 376, 556
larsen, E. G., 967, 1178
larson, F. C., 1079, 1173
lasagna, L., 1468, 1466
Laskowski, M., 1081, 1052
IaSorsa, A. M., 1098, 806
laszlo, D., 570, 110; 1328, 1482
latterell, F. M., 1046, 930
lauderdale, V., 303, 686
Laughton, R., 235, 757; 1589, 1641
laurent, D., 342, 769
lauson, H. D., 993S
lavenda, N., 377, 772
lavik, P. S., 876, 51
lawton, R. W., 378, 432
layne, E. C., Jr., 968, 1148
lagzarini, R., 787, 1213
leak, J. C., 883, 1802
leake, C. D., 1370, 1672
Leake, N. H., 1456, 1441
leary, D. E., 1359, 1573
leathem, J. H., 444, 412
Leaver, F. W., 969, 1089
Lebow, M., 186, 424

leBrie, S. J., 30, 517

Leder, I. G., 970, 1369
Ledford, E. 8., 1742, 1748
Lee, J. M., 1824, 1896

lee, K. H., 805, 1151

Lees, M., 973, 997
leftwich, C. I., 216, 684
Lehman, I. R., 950, 1185
Lehninger, A. L., 765, 1015
lehotzky, H., 1400, 1418

Lehr, D., 1469, 65; 1470, 1682; 1485, 64
leichsenring, J., 1769, 1909; 1874, 1908
leifer, P., 1396, 1622

Lemon, H. M., 1101, 55

Leon, M. A., 1695, 2063

Leone, C. A., 1950, 2024

Leong, G. F., 1817, 1650
Leoschke, W. L., 1825, 1858
LePage, G. A., 1060, 985
Lerner, A. M., 2011, 1945
LeRoy, G. V., 1511, 108
Leskowitz, S., 1951, 160

Lessler, M. A., 266, 101

lester, G., 380, 315

lester, R. L., 974, 1016

lettvin, J. Y., 328, 470
Leuchtenberger, C., 1696, 1753
Levedahl, B. H., 381, 1047
Levenberg, B., 382, 914
Leveque, P. E., 1471, 1413
Leverett, S. D., Jr., 521, 576a
levine, L., 732, 1112; 1952, 2023
levine, V. E., 975, 1370
Levintow, L., 976, 1297

Levitz, M., 977, 1267

levy, H. R., 978, 818

Levy, J., 906, 986

AUTHOR INDEX

Levy, M., 979, 969

Levy, M. N., 384, 434
Lewbart, M. L., 980, 1355
Lewin, R., 1116, 1129
Lewis, C., 991, 1211

Lewis, G. T., 981, 1106
Lewis, J. H., 154, 151
Lewis, L. A., 385, 80

Lewis, L. J., 1992, 1950
Lewis, M. S., 386, 259
Lewis, N. M., 1472, 1425
Lewis, W. J., 26, 461
Lewycka, C., 784, 1066

Li, K., 1953, 1986
Lichtenstein, J., 864, 1117
Lichtler, E. J., 221, 79
Lichtman, H. C., 808, 1785
Lieberman, I., 982, 1120
Lieberman, M., 1915, 1940
Lifson, N., 412, 197

Light, A. E., 1826, 1894
Lilienthal, J. L., Jr., 282, 554
Lilly, E. M., 862, 1367
Lilly, J. C., 387, 324; 388, 773
Lim, R. K. 8., 1500, 1566
Lin, T. M., 389, 2

Lind, H. E., 1306, 1554
Lindberg, O., 726, 1013
Lindberg, R. B., 1953, 1986
Lindeman, V. F., 390, 774
Lindsey, A. W., 271, 425
Liner, R., 842, 1240

Ling, G., 391, 246
Linkenheimer, W. H., 392, 624
Linkswiler, H., 1769, 1909; 1874, 1908
Lionetti, F. J., 983, 1123
Lipke, H., 984, 966
Lipmann, F., 773, 1299; 1105, 1022
Lippincott, S. W., 1697, 139
Liptak, R. A., 280, 494
Littauer, U. Z., 985, 1258
Little, J. M., 1473, 1628
Littlefield, J. W., 986, 879
Littlefield, S., 1333, 1464
Liu, C., 1954, 1999

Loeb, G. I., 1138, 970
Loeser, C., 1724, 1046
Loew, E. R., 1517, 1565
Loewe, 8., 1420S

Loewus, F. A., 987, 924
Logan, M. A., 1055, 896
Logan, R. G., 20, 730
LoGrippo, G. A., 1682, 1720; 1955, 1973
Lojkin, M. E., 1827, 1932
London, I. M., 992, 622
Long, N. J., 1875, 1841
Longley, R. W., 988, 1061
Longson, D., 323, 405
Longwell, B. B., 989, 1072
Loomis, T. A., 1474, 149
Loomis, W. F., 990, 647
Loosli, C. G., 1928, 2011
Loosli, J. K., 1877, 1807
Lopez-Mendoza, E., 292, 763
Lorand, L., 393, 263

Lord, G., 1513, 90

Loring, J. M., 1228, 1290
Losee, F. L., 1686, 1704
Losner, 8., 1743, 114
Lotspeich, W. D., 394, 290
Lotz, L. V., 862, 1367
Loud, A. V., 395, 845
Love, R., 1946

Love, R. A., 1666, 1735

Loveless, M. H., 1956, 2041
Lowe, C., 1698, 836

Lowell, F. C., 1982, 1983
Lowry, O. H., 991, 1211
Lowy, B. A., 992,
Lozaityte, I., 1012, 1198

Lu, Go, 1400, 1418; 1574, 1535
Lucchina, G. G., 103, 602
Luck, J. M., 990S

Ludwig, E. H., 1957, 1976
Ludwig, M. L., 1026, 1142
Lukens, L. N., 993, 913
Lun, G. §., 1475, 1550
Luper, M., 914, 1136

Lurie, M. B., 1958, 2053
Lushbaugh, C. C., 1699, 73
Lushbough, C. H., 1828, 1812
Lutz, B. R., 51,M.P., p. 628; 52, 735
Lutz, R. N., 1829, 1917
Luyet, B. J., 223, 625
Lyman, M. M., 1849, 61
Lynen, F., 953, 1227
Lynes, T. E., 1305,M P, p. 628; 1419, 1660
Lynn, W. §., Jr., 994, 1352
Lyon, J. B., Jr., 396, 462

Maas, W. K., 995, 1216

Maass, A. R., 1430, 1578

MacCanon, D. M., 1417, 1402

MacDonald, M. A., 369, 645

MacGrath, W. B., 23, 387

Machlin, L. J., 996, 1143

Macht, D. I., 397, 321; 1476, 1683

Mackal, R. P., 1053, 1188

Mackenzie, C. G., 997, 1734

Mackenzie, J. B., 997, 1734

Mackler, B., 810, 1235

MacLean, L. D., 398, 676

MacLeod, R. A., 998, 934

MacLennan, J. D., 1931, 2043

Macmillan, W. H., 399, 441; 1580,1563

Madden, D., 1640, 1702

Madden, R. J., 400, 707

Maddock, 8., 1700, 1706

Magalini, S. I., 401, 116

Magasanik, B., 1038, 1118

Magee, D. F., 402, 702

Magid, E. B., 321, 699

Magladery, J. W., 604, 468

Maher, F, T., 1701, 1762

Mahler, H. R., 999, 816

Mahoney, D. C., 1598, 1544

Mahowald, T. A., 1000, 851

Maire, F. W., 403, 267

Major, C. W., 610, 457

Makovsky, A., 1551, 1611

Malamed, S., 404, 693

Malament, S. 792, 236

Maley, G. F. 1001, 1323

Maling, H. M. 1346, 284; 1477, 1414; 1478,
1684

Malvin, R. L., 405, 296

Mandel, H. G. 1002, 1261; 1478, 1684

Mangay, A. S., 1830, 1933

Mangel, M., 1769, 1909

Mann, F. D., 1702, 38

Mann, G. V. 1763, 8; 1790, 10; 1831, 6

Manning, J. W., Jr., 276, 23

Mansmann, H. C., Jr., 2023, 2049

Mansor, L. F., 1337, 1536; 1479, 1534

Mansour, J. M., 1320, 1474

Mansour, T. E., 1480, 1509

xii

Mantel, N., 1423S

Manthei, R. W., 1314, 1462; 1481, 1685

Manuelidis, E. E., 1978, 2040

Marco, G. J., 1003, 1285

Marcus, 8., 1959, 157

Maren, T. H., 1402, 1540; 1576, 1541

Margolin, S., 1586, 1617; 1607, 1499

Margulies, M., 1004, 942

Marinetti, G. V., 1005, 1000; 1264, 999

Mark, L. C., 1482, 1389

Markowitz, J., 102, 523

Markus, G., 1960, 975

Marmorston, J., 1997, 449

Maroney, 8. P., Jr., 406, 167

Marrazzi, A. S., 281, 376; 1467, 1574

Marsh, D. F., 1483, 1592

Marsh, J. B., 1006, 1283

Marsh, M. E., 1832, 1934

Marshall, L. M., 1007, 1220

Marshall, N. B., 1835, 1802

Marshall, W. H., 188, 534

Martel, A., 1732, 1782

Martin, A. W., 407, 638

Martin, C., 1469, 65; 1470, 1682; 1485, 64

Martin, C. J., 665, 354

Martin, Charles J., 1484, 1508

Martin, F. B., 1089, 1007

Martin, G. J., 705, 1790

Martin, 8. J., 44, 733

Martin, W. R., 1355, 1571; 1486, 1567

Martin, William R., 1008, 1371

Martin, W. S., 266, 101

Martinez, W. H., 1039, 1033

Marusich, W., 1833, 1877

Marx, L. 831, 145

Marx, W. 831, 145

Masin, F., 1748, 1731

Mason, B. T., 1961, 2073

Mason, H. C., 1961, 2073; 1962, 2074

Mason, H. S., 1009, 1229; 1200, 1159

Mason, M., 1010, 1031

Mason, R. B., 415, 238

Mason, R. C., 1487, 1547

Masoro, E. J., 194, 1292; 408, 539

Massey, V., 932, 1233; 1011, 1234

Masson, G. M. C., 409, 566; 1354, 1583

Masuda, M., 410, 407

Mathews, M. B., 1012, 1198

Matschiner, J. T., 903, 852

Mattson, F. H., 1760, 1836; 1834, 1296

Maurer, P. H., 1963, 2022; 2026, 2065

Maxwell, H. R., 1918, 2058

Maxwell, R. A., 1488, 1526; 1522, 1658

Maxwell, R. E., 1489, 1406

May, K. J., 2028, 1943

Mayer, J., 670, 47; 1835, 1802

Mayer, M: M., 1964, 1974

Mazurkiewicz, I., 899, 1472

Mazzocco, T. R., 623, 795

McAdams, A. J., 1688, 1779

McAllister, J. G., III, 1746, 113

McArdle, A. H., 1362, 1618

McArthur, J. W. 686, 1271

McCallum, G. L., 1965, 2070

McCandless, E. L., 669, 57

McCann, M. B., 1878, 1918

McCann, S. M., 12,M.P., p. 628

McCarthy, M.D., 411, 671

McCarty, B. 910, 21

McCay, P., 1013, 1277

McClendon, J. F., 205, 460; 1014, 1372;
1836, 1915

McClintock, R., 412, 197

McColl, J. D., 924, 97

FEDERATION PROCEEDINGS

McCollum, E. B., 1814, 1826

McConahey, W. M., 454, 779

McConn, R. G., 1341, 1604

McConnell, K. P., 1015, 1373

McCormick, W. G., 1409, 1676; 1410, 1607

McCoy, T. A., 1016, 18

McCulloch, W. S., 328, 470

McCullough, W. G., 1664, 1289

McCutcheon, R. S., 1490, 1564

McDaniel, E. G., 1024, 1055

McDonald, H. J., 715, 1361

McDonald, R. K., 413, 518

McDuffie, F. C., 1966, 2042

McElroy, W. D., 1017, 960

McGaughey, C., 817, 1113

McGill, H. C., Jr., 1676, 1744; 1735, 1

McGinnis, J., 1818, 1897

Mcllreath, F., 264, 146

McIntyre, A. R., 1491, 1519

MclIsaac, R. J., 1492, 1646

McKee, R. W., 910, 21

McKenna, J. M., 673, 673; 2008, 2005

McKennis, H., Jr., 1493, 1390

McKey, B. V., 1870, 1903

McLain, P. L., 1494, 1686

McLaughlin, M., 478, 231

McLean, I. W., Jr., 1967, 1948; 2013, 1952;
2016, 1951

McLean, J. R., 1018, 881

McLellan, W. L., 983, 1123

McManus, I. R., 1019, 1248

MeMillan, A., 1155, 1171

McMillan, M., 1132, 1919; 1870, 1903

McMurray, W. C., 1020, 1004

McNamara, H., 363, 453

McNerney, J. M., 1678, 1768

McOsker, D. E., 1021, 168

McQuillen, M. P., 656, 420

McRorie, R. A., 1022, 1246

McVaugh, R. B., 325, 561

McVickar, D. L., 1968, 2068

Mead, J., 414, 228

Mead, J. F., 179S; 1023, 1293

Meath, J. A., 826, 996

Meckelnburg, K. L. 1590, 1429

Megirian, R., 1495, 1500

Mehl, J. W., 1183, 1251

Mehler, A. H., 1024, 1055

Meier, R. M., 288, 666

Meisel, E., 1745, 1761

Meister, A., 976, 1297; 1088, 1301

Melampy, R. M., 415, 238

Melchior, J. B., 1025, 1090

Melchior, N. C., 1025, 1090

Mellors, R. C., 1703, 1757; 1715, 1756

Melnick, I., 382, 914

Melnick, J. L., 1895, 1954; 1932, 1972;
1985, 1970

Meltzer, H. L., 416, 1343

Melville, D. B., 1026, 1142

Melville, G. S., Jr., 1663, 1793

Melville, K. I., 1461, 1410

Mendicino, J., 1027, 1024

Meng, H. C., 158, 663; 305, 89; 553, 152

Menkin, V., 417, 587

Meriwether, B. P., 459, 1150

Merrick, A. W., 418, 775

Merrifield, R. B., 1028, 1107

Mertz, E. T. 1029, 119; 1768, 1860; 1777,
1862; 1782, 1864

Mertz, W., 1837, 1878

Merwin, R. M., 419, 165

Metta, V. C., 177S

Meyer, D. K., 418, 775

Volumett J
Meyer, E. D., 836, 1359 M
Meyer, F., 1083, 1376 M
Meyer, H. M., Jr., 1969, 1975 M
Meyer, J. S., 1683, 1774 M
Meyer, K., 886, 1202 M
Meyers, F. H. 1581, 1391 M
Meyers, R. E., 436, 777 M
Meyeserian, M., 1942, 2004 M
Michaelson, 8S. M., 420, 138 M
Michel, H. O., 1030, 821 M
Mickelsen, O., 1887, 1892 M
Middo, R. T., 463, 209 M
Mielke, J. E., 1705, 1767 M
Mihajlov, V., 177, 751 M
Mika, E. 8., 1620, 1613 M
Milch, L. J., 1538, 1428 M
Milhorat, A. T., 1882, 1876 Mi
Millar, G. J., 421, 44 Mi
Miller, A., 1031, 1332
Miller, A. T., Jr., 422, 465 Mc
Miller, B. F. 827, 444 M
Miller, B. J., 1927, 1993 Mc
Miller, C. P., 1712, 1711 Mc
Miller, H. I., 573, 86 Mc
Miller, L. L., 740, 886 Mc
Miller, L. T., 1913, 2071 Mc
Miller, M., 132, 196 Mc
Miller, O. N., 1223, 1920; 1799, 1814 Mc
Miller, R. F., 1838, 1882 Mr
Miller, S. A., 1761, 1905 M
Miller, S. L., 1032, 1036 M
Millican, R. C., 1496, 1595 M
Millman, N., 423, 239 Mu
Mills, J. N., 1167, 859 Mu
Mills, R. C., 1232, 1237 Mi
Milne, W. L., 1339, 107; 1704, 30 Mu
Milnor, W. R., 424, 495 Mu
Milora, R., 1469, 65; 1470, 1682 Mu
Minard, D., 45, 685; 425, 636 Mu
Minatoya, H., 1498, 1594 Mu
Minthorn, M. L., 947, 1318 Mu
Minton, M. F., 113, 505; 671, 504 Mu
Minz, B., 1497, 1687 Mu
Misko, J. C., 1523, 1690 My

Mistry, 8S. P., 1033, 918
Mitchell, 8. Q., 1499, 1417
Mitz, M. A. 1970, 1340

Miya, T. 8., 1345, 1451 N
Miyake, A., 1083, 1376

Moat, A. G., 1971, 917 Nai
Moffit, R. L., 1500, 1566 Nay
Mogab, J. J., 1988, 2056 Nal
Moldave, K., 426, 49 Naj
Moll, H. C., 427, 607 Nal
Monagle, J. E., 428, 505a Nai
Mone, P. E., 1862, 1805 Nal
Monier, J. 8., 1626, 188 Nas
Monkhouse, F. C., 429, 144 Nas
Monroe, C., 1640, 1702 Nas
Moor, J. R., 1880, 1925 Nas
Moore, D. H., 1972, 2016; 1986, 2015 Nat
Moore, G. M., 1317, 1650 Nat
Moore, J. C., 430, 226 Nat
Moore, R., 1501, 1528 Nea
Moore, R. O., 1095, 1077 Nef
Moore, S., 874S; 1173, 1339 Nei
Moos, C., 393, 263 Neil

Morales, G., 1502, 1490; 1514, 1467; 1637, 8 Nel;

1610
Morales, M. F., 63, 261; 431, 957 Nel:
Morales, S., 1883, 1916 Nel:
Moran, N. C., 1477, 1414; 1503, 1452; 1529, § Nel:
1453 Nels
Morehouse, M. G., 1034, 142 Nel:

lume Ij

1814

2015

(467; 1637,

1452; 1529,

March 1956

Morello, A., 1502, 1490

Morgan, A. F., 1839, 1935

Morgan, C., 1972, 2016; 1986, 2015

Morgan, E. R., 963, 939

Morgan, J. F. 1035, 805

Morgan, P., 1750, 1771

Morgan, W. L., 59, 210; 432, 211

Mori, H. D., 1706, 1781

Morin, F., 433, 776

Morphis, B. B., 1552, 1600

Morrill, G. A. 789, 1475

Morrison, A. B., 1840, 1886

Morrison, M., 764, 813; 1036, 1228

Morrow, G., 28, 718

Morton, H. J. 1035, 805

Morton, J. I., 788, 1194

Morton, J. L., 1649, 24

Mosbach, E. H., 1323, 954; 1647, 1778;
1707, 1777

Moskowitz, M., 1984, 1984

Moss, W. G., 495, 577

Motley, H. L., 216, 684

Mounter, L. A., 1037, 965

Moy, R. H., 1753, 12

Moyed, H. S., 1038, 1118

Moyer, J. H., 1347, 1581; 1450, 1586

Mozden, P., 1517, 1565

Mozersky, 8., 920, 809

Mraz, F. R., 1841, 1810; 1857, 1880

Mueller, G. C., 893, 1269; 1638, 1648

Muirhead, E. E., 1708, 1797a

Mukherjee, S., 1776, 1851

Muldrey, J. E., 1039, 1033

Mulford, D. J., 1040, 1374

Miiller, O. H., 434, 889; 639, 342

Munch-Petersen, A., 697, 941

Murayama, M., 1041, 812

Murdoch, M. E., 1316, 1338a

Murphy, 8. D., 1504, 1688

Murray, I. M., 1042, 977

Murray, T. K., 1842, 1840

Musacchia, X. J., 435, 479

Mushett, C. W., 1709, 67; 1876, 9

Myrvik, Q. N., 1973, 2054

Naael, E. M., 748, 1071; 1710, 1798; 1711,
1727

Nadell, J., 171, 708

Nagasawa, H. T., 868, 94

Nahas, G. G., 437, 578

Najjar, V. A., 1162, 1094

Nakada, H. I., 438, 901

Nanninga, L., 1043, 842

Nardone, R. M., 439, 589

Nash, J. B., 1505, 1386; 1636, 1549

Nason, A., 1174, 856

Nasset, E. S., 1843, 1907

Nastuk, W. L., 440, 191

Nathan, P., 14, 325

Nations, F. N., 1624, 1701

Natvig, R., 1044, 1100

Neal, A. L., 1107, 1884

Neff, J. C., 1974, 2021

Neidle, A., 1126, 878

Neilands, J. B., 1045, 1312

Nelson, A. A., 1315, 1404; 1373, 1405;
1757, 1783

Nelson, C. T., 175, 317

Nelson, E., 303, 686

Nelson, E. L., 1712, 1711; 2027, 2038

Nelson, L., 1737, 1776

Nelson, M. M., 1787, 1817; 1844, 1827

AUTHOR INDEX

Nelson, T. L., 1521, 1689
Nelson, T. S., 1867, 1871
Neufeld, H. A., 1046, 930
Neuhaus, O. W., 1047, 804
Neuman, R. E., 1016, 18
Neuman, W. F., 1195, 1133
Neurath, H., 933, 895
Newsom, B. D., 441, 508
Newton, G., 713, 1078
Neyland, M., 204, 527
Nichol, C. A., 1275, 1333
Nicholas, A., 1937, 2017
Nicholas, H. J., 1048, 844
Nichols, C. W., Jr., 1856, 4
Nichols, G., Jr., 1845, 199
Nichols, J., 442, 595
Nichols, N., 1845, 199
Nichols, N. H., 403S
Nickerson, M., 1506, 1602; 1570, 1577;
1634, 1597
Niden, A. H., 16, 351
Nielsen, F. J., 2022, 849
Ninos, G. 8., 1958, 2053
Nisonoff, A., 1049, 1959
Nisselbaum, J. 8., 716, 1346
Nitowsky, H. M., 1846, 1874
Noble, F. W., 443, 286
Noble, N. L., 337, 1201; 1507, 91
Noble, R. L., 97, 5
Nocenti, M. R., 444, 412
Noda, L., 1050, 1026
Noe, E., 287, 123
Noell, W. K., 445, 690
Nolan, J. M., 1902, 2055
Nold, M. M., 1881, 1865
Norcia, L. N., 839, 66
Nord, F. F., 1210, 887
Norman, D., 446, 778
Norman, P. 8., 1975, 120
Normann, N., 447, 688
Norris, A. H., 448, 229
Norris, L. C., 1840, 1886
North, W. C., 1452, 1596
Northup, D. W., 585, 705
Norton, P., 1871, 1861
Norton, S., 1508, 1655
Nossal, P. M., 1051, 1017
Nothman, M. M., 1509, 19
Novack, P., 371, 207
Novelli, G. D., 785, 875
Novikoff, A. B., 1052, 983
Nowinski, W. W., 449, 716
Noyes, W. F., 1713, 1751
Nungesser, W. C., 450, 480
Nussbaum, A., 588, 1291
Nye, J. F. 1069, 1825
Nye, S. W., 227, 756a
Nyman, M. A., 278, 81

Onrien, L. J., 451, 208

Ochoa, 8., 873S; 734, 1255; 891, 1256
O’Dell, B. L., 1847, 1822

Odland, L. M., 1848, 1923
O’Donnell, J. F., 1053, 1188

Oester, Y. T., 1510, 63; 1550, 62
Oesterling, M. J., 1054, 1157
Ogburn, C. A., 1929, 2008

Ogle, J. D., 1055, 896

O’Halloran, P. 8., 75, 224

Ohlson, M. A., 1805, 1912; 1821, 1913
Ohno, S., 1714, 133

Oikemus, A. H., 1512, 1435

Okey, R., 1849, 61

Okita, G. T., 1511, 108

Okuda, K., 1814, 1826; 1850, 1823

Olds, J., 1454, 1652

O’Leary, J. F., 1512, 1435

Oleksyshyn, N. L., 187, 860

Oleson, J. J., 1218, 98

Olewine, D. A., 467, 780

Olin, C. I., 504, 784

Olitsky, P. K., 1824, 1896

Oliver, R. W., 1802, 1842

Olsen, N. 8S., 1057, 998

Olson, J. A., 1056, 847

Olson, M. E., 1085, 883

Olson, R. E., 470, 205; 1188, 1103; 1888,
1821

Olwin, J. H., 949, 34; 1642, 39

Omachi, A., 233, 618

O'Malley, W. E., 1321, 1599

Oncley, J. L., 365, 1348; 853, 84

Oneson, I., 1513, 90

Ono, J., 1754, 1723

Onofrey, E., 998, 934

Onoprienko, I., 1009, 1229

Oppenheimer, F., 2013, 1952

Orbison, J. L., 1724, 1046

Orkin, L. R., 1514, 1467; 1637, 1610

Orloff, J., 452, 293

Ormsby, A. A., 596, 222

Ortega, L. G., 1703, 1757; 1715, 1756

Orten, A. U., 1851, 1856

| N., 347, 770; 609, 525

Paganelli, C. V., 456, 617

Page, A. C., 1786, 1889

Page, A. C., Jr., 1852, 1890

Page, E., 706, 1214

Page, I. H., 385, 80

Paldino, R. L., 457, 410

Palleroni, N. J., 794, 950

Pallotta, A. J., 1516, 1626

Panagos, S. 8., 194, 1292

Panos, T. C., 458, 237

Papacostas, C. A., 1517, 1565

Papadopoulos, N. M., 1853, 1804

Pappenheimer, A. M. Jr., 2018, 2028;
2033, 2048

Papper, E. M., 1482, 1389

Pareira, M. D. 1977, 678

Parfentjev, I. A., 1978, 2040

Park, C. R., 459, 1150; 1058, 1062

Park, J. H., 459, 1150

XIV

Parker, R. E., 1594, 1542
Parlow, A. F., 30, 517
Parsons, H. T., 1132, 1919
Parsons, J., 1270, 115

Partin, H. C., 1507, 91
Partridge, M. H., 449, 716
Paschkis, K. E., 94, 1260; 460, 408
Pasik, P., 461, 417

Pasik, T., 461, 417

Passey, R. F., 1059, 909
Passonneau, J. V., 778, 1241
Patek, A. J., 1904, 1910
Paterson, A. R. P., 1060, 985
Patrick, H., 1841, 1810; 1857, 1880
Patt, H. M., 589, 132
Patterson, E. K., 1061, 894
Patterson, J. L., Jr., 198, 273
Patton, F., 1070, 1770
Paulson, 8. F., 668, 701
Payza, A. N., 1062, 1196
Pazur, J. H., 1063, 952

Pearl, D. C., Jr., 462, 262
Pearl, W., 263, 640

Pearson, H. E., 1979, 1995
Pearson, N. S., 1880, 1925
Pearson, P. B., 700, 1021; 1823, 1881
Pearson, W. N., 1830, 1933; 1854, 1816
Peck, H. D., Jr., 1064, 958
Pederson, D. J., 688, 1833
Peek, N. F. 1856, 4

Peifer, J. J., 1065, 1853

Peiss, C. N., 349, 304; 463, 209
Penek, M. J., 1574, 1535
Penn, H. 8., 1543, 128

Penn, N., 1980, 2067
Pennell, R. B., 686, 1271
Penniall, R., 1981, 1147
Penrod, K. E., 464, 695
Perdue, H. 8., 1855, 1855
Pérez, C., 1868, 1921
Perkins, J. F., Jr., 465, 302
Perl, E. R., 466, 473

Perlin, S., 565, 372

Perlmutt, J. H., 467, 780
Perot, P. L., Jr., 468, 692
Petermann, M. L., 1066, 1042
Peters, J. H. 868, 94

Peters, T., 1982, 1983
Petersen, D. F., 1518, 74
Petersen, L. H., 469, 429
Peterson, D. W., 1856, 4
Peterson, R. D., 1067, 1789
Phillips, G. E., 792, 236
Piantadosi, C., 1068, 1375
Piatnek, D. A., 470, 205
Pick, R., 578; 792

Pierce, J. G. 1069, 1325
Pierce, W. C., 747, 1109
Pierce, Z. H., 1829, 1917
Pietroluongo, A. L., 400, 707
Piez, K. A., 1519, 1040
Pigman, W., 1070, 1770

Pike, R. L., 1863, 1830
Pillemer, L., 1913, 2071; 2025, 2069
Pinajian, J. J., 1857, 1880
Pincus, G., 667, 411; 722, 1070
Piovella, C., 1897,M P, p. 628
Pipkin, G. E., 1885, 1895
Pirrung, J., 772, 1172

Pitt, B. M., 1071, 964
Pittinger, C. B., 1520, 1471
Pitts, R. F., 351, 343

Pitts, W., 328, 470

Plaa, G. L., 1521, 1689

FEDERATION PROCEEDINGS

Platner, W. S., 471, 781

Platt, D., 1070, 1770

Plescia, O. J., 440, 191; 1983, 2066

Pless, H. H., 1302, 1657

Plotnikoff, N. P., 1283, 1561

Plummer, A. J., 1352, 1436; 1488, 1526;
1522, 1658; 1571, 1529; 1603, 1576

Podber, E., 1061, 894

Pogrund, R. S., 472, 653

Polglase, W. J., 473, 1209

Poling, C. E., 1862, 1805

Polis, B. D., 1072, 1014

Pollack, R. L., 1073, 922

Pollard, C. J., 1767, 1850

Polley, E., 1614, 1699

Pomerantz, S. H., 1074, 1088

Poppell, J. W., 499, 496

Popper, H., 1716, 1772

Porrata-Doria, E. I., 1779, 1927

Porter, G. A., 1523, 1690

Porter, J. C., 474, 365

Porter, J. W. 1234, 1286

Porter, R., 540, 344

Porter, T., 1828, 1812

Portman, O. W., 1858, 7

Post, R. L., 475, 445

Postel, S., 1722, 1730

Potter, J. L., 1075, 838

Potter, V. R., 882, 1184

Powell, J. R., 1810, 1835

Pozza, G., 203, 755

Pratt, J. H., 1509, 19

Pratt, R. L., 660, 633

Prediger, F. R., 1349, 1580

Pressman, B. C., 1076, 1012

Pressman, D., 1049, 1959; 1896, 2001

Preston, J. B., 1524, 1572

Price, C. A., 1077, 1338

Price, H. L., 1525, 1557

Price, D. B., 1078, 885

Price, J. M., 733, 1176; 1079, 1173

Price, N. O., 1838, 1882

Pricer, W. E., Jr., 1086, 864

Prioli, N. A., 1526, 1601

Pritchard, E. T., 1080, 1005

Privat de Garilhe, M., 1081, 1052

Procita, L., 1426, 1459; 1527, 1458

Proctor, C. D., 1528, 1892; 1533, 1518;
1534, 1517

Prosser, C. L., 401S

Proutt, L. M., 89, 371

Prusoff, W. H., 1082, 1182

Pruzansky, J., 1859, 1819

Pryor, J. W., 2024, 1996

Pudelkewicz, C., 1860, 1818

Purdy, F. A., 1622, 1700

Purpura, D. P., 476, 377

Putnam, F. W., 1083, 1376

ER G. G., 1821, 1913
Quick, A. J., 1084, 147

Quilliam, T. A., 477, 422

Quinn, G. P., 1503, 1452; 1529, 1453
Quiroz, A., 1883, 1916

Tue, G., 243, 285
Rabiner, 8. F., 808, 1785
Rabinovitz, M., 1085, 883
Rabinowitz, J. C., 1086, 864
Rachele, J. R., 1087, 1250

Volume 16

Racker, E., 845, 1008

Radding, C. M., 1235, 962

Radford, E. P., Jr., 478, 231

Radhakrishnan, A. N. 1088, 1301

Radin, N. S., 124, 217; 1089, 1007

Rafter, G. W., 1090, 833

Rafter, J. 961, 109

Rahn, H., 479. 583

Rajou, R. A., 1668, 1759

Rall, D. P., 1530, 1627

Rall, T., 1091, 1155

Ralli, E. P., 480, 299

RamaRao, P. B., 1033, 918

Ramey, E. R., 55, 458; 247, 361

Rampone, A. J., 196, 725

Ramsey, D. S., 1924, 1981

Rand, R., 1698, 836

Randall, C. C., 1€51, 1722; 1717, 1721

Randall, H. T., 499, 496; 620, 497

Randall, J. E., 481, 430; 1555, 1694

Randall, L. O., 1343, 1524; 1567, 1623

Randall, W. C., 463, 209; 506, 486

Rapp, H. J., 1964, 1974

Rappaport, A. M., 1092, 447

Rappoport, D. A., 1531, 76

Rapport, D., 95, 111; 194, 1292

Rapport, M. M., 1093, 1960; 1713, 1751

Ratliff, F., 482, 528

Rau, G. C., 33, 732; 1532, 1614

Rauschkolb, E. W., 191, 362

Raut, C., 1094, 1207

Rawnsley, H. M., 752, 1210

Ray, B. R., 984, 966

Ray, F. E., 1718, 613

Read, K., 1984, 1984

Read, M. S., 184S; 1095, 1077

Reazin, G. H., 1096, 906

Rebar, B. T., 1528, 1892; 1533, 1518; 1534,
1517

Rebar, J. Jr., 1528, 1392; 1533, 1518; 1534,
1517

Rebuck, J. W., 1719, 1708

Reckers, L., 1097, 1272

Recknagel, R. O., 483, 1167

Reeb, B. B., 124, 217

Reed, E. A., 484, 227

Rehm, W. S., 148, 610

Reichard, S. M., 485, 665

Reid, B. L., 1099, 1885

Reid, E., 529, 788

Reid, M. E., 1861, 1906

Reid, 8. C., 1427, 1608

Reif, A. E., 989, 1072

Reiner, B., 486, 1130

Reiner, J. M., 486, 1130

Reinhardt, W. O., 487, 782

Reinhold, J. G., 752, 1210

Reiser, R., 1100, 1294

Reissig, M., 1985, 1970

Relman, A. S., 488, 345; 540, 344

Remp, D. G., 1924, 1981

Rendell-Baker, L., 1535, 1531

Rennick, B. R., 1536, 1590

Renold, A. E., 217, 588

Renzi, A. A., 1538, 1428

Repplinger, E. F., 1537, 1691

Reynolds, M. D. 1101, 55

Reynolds, M. S., 1769, 1909; 1774, 1815

Reynolds, V. H., 1216, 945

Rhode, R., 172, 630

Rhodes, D. N., 870, 1001

Rhodes, M. B., 813, 940

Ricci, G., 489, 603

ume 1§

, 1751

18; 1534,

1815

March 1956

Rice, E. E., 1862, 1805
Rich, C. 1102, 1309

Rich, K., 1276, 1183

Rich, M., 236, 609; 1589, 1641
Richards, A. B., 1387, 1523
Richards, G. E., 1146, 1050
Richards, J. B., 490, 367
Richards, R. K., 1539, 1463; 1597, 1537
Richardson, D. W., 198, 273
Richardson, E. M., 611, 592
Richardson, J. A., 1540, 1558
Richardson, L. R., 181S
Richert, D. A., 895, 1883; 1144, 1179
Richter, M., 542, 974

Rieck, A. F., 491, 140

Rieder, S. V., 1103, 1097
Rieders, F., 1541, 1401; 1542, 1692
Riedesel, M. L., 492, 545

Riegel, C., 533, 383

Rieker, R. A., 1178, 1888

Rieser, P., 493, 102

Rigdon, R. H., 1789, 1872

Riggs, T. R., 1104, 1137
Rightsel, W. A., 1967, 1948

Riley, R. F., 1543, 128

Riley, V. T., 494, 616

Rinaldi, F., 1898, 2051

Rinehart, J. F., 865, 1932; 1832, 1934
Ring, G. C., 495, 577

Ringer, R. K., 591, 560; 642, 798
Ringler, I., 496, 241

Rink, R. A., 1397, 1442
Rittenberg, D., 954, 959
Ritterband, A. B., 36, 218
Robbins, J., 497, 243

Robbins, M. L., 1464, 1551
Robbins, P. W., 1105, 1022
Roberts, E., 900, 860

Roberts, J. C., 2081S

Roberts, J. T., 498, 382

Roberts, K. E., 499, 496; 620, 497
Roberts, N. R., 991, 1211
Roberts, S., 561, 364

Roberts, W. J., Jr., 354, 661
Robertson, H. E., 1919, 1969
Robertson, J. D., 407, 638
Robertson, J. S., 500, 111a
Robertson, W. V., 1106, 1203
Robinson, C. W. 839, 66
Robinson, H. J., 1487, 1547
Robinson, M. J., 1375, 1553
Robinson, P. F., 501, 714
Robinson, R. J., 1499, 1417
Robinson, R. U., 1437, 1640
Robinson, T., 1107, 1884
Robinson, W. G., 1108, 1083
Roboz, E., 938, 1724

Rockstein, M., 502, 783
Rodbard, S., 503, 287; 504, 784
Roderuck, C., 1860, 1818
Rodkey, F. L., 1109, 1085
Rodnan, G. P., 505, 639

Rodwell, V. W., 1217, 1215

Roe, J. H., 988, 1061; 1853, 1804
Roeder, K. D., 612, 598

Rogers, D., 1110, 938

Rogers, E. B., 11, 567

Rogers, N. G., 1969, 1975
Rohrbaugh, L. M., 1213, 944
Rohse, W. G., 506, 486

Rokaw, 8. N., 24, 502

Roley, E. L., 75, 224

Rolfe, D. T., 87, 1824

Roma, M. 803, 555

E XUM

AUTHOR INDEX

Rondell, P. A., 325, 561
Rongone, E. L., 1111, 1168
Roos, T. B., 658, 629
Root, M. A., 1544, 1645
Rope, E. Z., 1967, 1948
Rose, B., 542, 974
Rose, C. L., 1545, 1396
Rose, C. S., 1112, 1204
Rose, H. M., 1972, 2016; 1986, 2015
Tose, I. A., 1103, 1097
Rose, N. R., 1987, 2020
Rose, W. C., 1866S
Rosebury, T., 1988, 2056
Roseman, 8. 1113, 1315
Rosen, E. A., 1532, 1614
Rosen, F., 423, 239
Rosen, H., 1546, 1642
Rosenberg, B., 750, 1048
Rosenberg, L., 1162, 1094
Rosenberg, P., 1547, 190
Rosenblith, W., 489, 603
Rosenfeld, B., 1092, 447
Rosenfeld, G., 507, 593
Rosenfeld, M., 1548, 117
Rosenfeld, R. S., 1114, 848
Rosenfeld, S., 1997, 449
Rosenthal, O., 1115, 1377
Rosenthal, S. M., 1201, 865; 1595, 1616a
Rosenthal, S. R., 508, 785; 1720, 1766
Roshe, J., 1684, 1716; 1721, 1717
Rosnagle, R. 8., 191, 362
Rosoff, B., 1116, 1129
Rosoff, M., 750, 1048
Ross, C. A., 1549, 1527
Ross, M. L., 1863, 1830
Ross, R. S., 636, 574
Ross, S., 1488, 1526
Rostorfer, H. H., 509, 814
Roszkowski, A. P., 1550, 62
Roth, D., 1015, 1373
Roth, F. E., 1551, 1611
Roth, J. S., 1117, 827
Roth, K. L., 1118, 1964
Roth, L. W., 1552, 1600
Rothman, §., 1553, 1448
Rothstein, A., 48, 717; 1136, 1091
Rouser, G., 1119, 811
Routh, J. I., 796, 1335
Rovenstine, E. A., 1554, 1693
Rowen, J. W., 1120, 1044
Rowen, R., 1989, 2057
Rowland, V., 510, 599
Rubin, S. H., 1833, 1877
Rubini, M. E., 511, 786
Ruch, T. C., 152, 423
Ruckle, G., 1990, 1997
Rudich, E. C., 491, 140
Rudman, D., 1991, 1782
Rudney, H., 1121, 1069
Rudoff, 8. L., 1122, 955
Rudolph, A. M., 24, 502
Rudolph, G. G., 512, 234
Rueckert, R. R., 1397, 1442
Ruegamer, W. R. 1123, 1822
Rumney, G., 1124, 1270
Rumsfeld, H. W., Jr., 474, 365; 1255,
808
Runge, R. R., 501, 714
Rusch, H. P., 1667, 935
Rushmer, R. F., 513, 281; 1621, 1454
Russell, J. A., 163, 713
Russell, P. B., 178, 752
Russell, R. L., 1555, 1694
Russfield, A. B., 1722, 1730

Russum, B. C., 522, 580
Rust, J., Jr., 1893, 2052
Ruth, H. J., 562, 161
Rutledge, L. T., Jr., 514, 467
Rutledge, R., 1522, 1658
Rutman, R. J., 493, 102
Ryan, A. H., 515, 1494
Ryan, K. J., 1125, 1162
Ryan, R. M., 324, 126

Sasa, F., 360, 260
Sabbagh, N. D., 1571, 1529
Sachs, B. A., 318, 459
Sachs, H., 1126, 878
Sacks, J., 516, 202
Sacktor, B., 517, 1152
Saito, A., 723, 1158
Salerno, P. R., 1556, 25; 1606, 75
Salgado, E., 1557, 83; 1558, 1695
Salhanick, H., 518, 1356
Salk, J. E., 1992, 1950
Sallach, H. J., 1127, 1140
Salmon, W. D., 180S; 1778, 14
Salter, J. M., 519, 712
Saltman, P., 1128, 1126
Salvador, R., 1129, 1145
Salvin, S. B., 2018, 2028
Salzano, J., 520, 507
Salzman, E. W., 521, 576a
Salzman, T., 1130, 1206
Samachson, J., 1171, 1134
Samaras, S. C., 522, 580
Samet, C., 1720, 1766
Sampson, M. M., 1864, 1936
Samson, F. E., Jr., 523, 370
Samuels, A. J., 1119, 811
Sanadi, D. R., 913, 1011
Sandberg, A. A., 1168, 1164
Sandow, A., 524, 787; 525, 258
Santer, U. V., 1131, 866
Santiago, L. S., 1132, 1919
Saphra, I., 672, 802
Sarber, R. W., 1993, 1953
Sarnoff, S. J., 59, 210; 71, 381; 99, 380; 432,
211; 577, 228; 586, 212
Saroff, H. A., 386, 259
Sather, G. E., 1927, 1993
Sauberlich, H. E., 180S; 1133, 1139
Saunders, D. H., 1692, 1797
Saunders, J. P., 1418, 18399; 1559, 1400
Savard, K., 1134, 1160
Sawyer, C. H., 219, 596
Saxton, G. A. Jr., 283, 352
Sayers, G., 526, 363
Saz, H. J., 1994, 1221
Scanu, A., 1143, 85
Scarpelli, D. G., 1744, 1754
Schachman, H. K., 1146, 1050
Schachter, D., 1560, 1395
Schachter, H., 274, 536
Schad, J. S., 1764, 1848
Schaechter, M., 1995, 1987
Schaefer, K. E., 527, 436
Schaffer, N. K., 1135, 820
Schambye, P., 1266, 951
Schanker, L. S., 1561, 1643
Scharff, T., 1136, 1091
Schayer, R. W., 1137, 389
Scher, A. M., 528, 489
Scheraga, H. A., 1138, 970
Scherf, D., 529, 788
Schiller, S., 1139, 1197

xvi

Schlegel, J. U., 177, 751; 530, 499

Schlenk, F., 1140, 862

Schlesinger, L., 644, 384

Schlumberger, H. G., 1723, 166

Schmatolla, E., 1904, 1910

Schmidt, C. F., 16, 351

Schmidt, G., 717, 1844; 1141, 1252

Schmidt, J. R., 1996, 1992

Schmidt, K. F., 1398, 1416

Schmidt, L. H., 1578, 1446

Schmidt-Nielsen, B., 531, 220

Schmidt-Nielsen, K., 532, 727

Schmitthenner, J. E., 533, 383

Schmolinske, A., 391, 246

Schneider, H. A., 1824, 1896

Schneider, J. A. 1501, 1528; 1603, 1576

Schneider, J. J., 980, 1355

Schneider, W. C. 1142, 16

Schneyer, C. A., 534, 706

Schneyer, L. H., 534, 706

Schoenberg, M. D., 1724, 1046

Schoene, R. B., 1794, 1839

Schoepfle, G. M., 535, 251

Schoffeniels, E., 536, 442

Schofield, R. A., 1644, 1715

Schinbaum, E., 537, 368

Schottelius, B. A., 538, 255

Schotz, M. C., 1143, 85

Schrack, W. D. Jr., 1927, 1993

Schraer, H., 1865, 1924

Schraer, R. 8., 1865, 1924

Schreiner, G., 47, 388

Schroeder, L. J., 1664, 1289; 1866, 1879

Schuck, C., 1817, 1863

Schueler, F. W., 1322, 171

Schulman, M. P., 1144, 1179

Schultz, F. H., Jr., 1594, 1542

Schultz, J., 1145, 973

Schulze, H. O. 696, 54

Schumaker, V. N., 1146, 105¢

Schwartz, H. 8., 1562, 1512

Schwartz, I. L., 319, 440; 320, 765

Schwartz, I. R., 1537, 1691

Schwartz, N. B., 539, 264

Schwartz, R., 1665, 1760

Schwartz, W. B., 488, 345; 540, 344

Schwarz, H. J., 737, 557

Schwarz, K., 754, 1278; 1837, 1878

Schweet, R., 1147, 880

Schweigert, B. S., 888, 1829; 1828, 1812

Schweiss, J. F., 1563, 1430

Schwerdt, C. E., 825, 1956

Schwert, G. W., 1148, 1098

Schwimmer, 8., 1149, 1378

Scott, C. C., 1334, 1637

Scott, JeG., 23, 387

Scott, M. L., 1840, 1886; 1867, 1871

Scott, W. W., 245, 438

Scriabine, A., 1564, 1484

Schrimshaw, N. S., 1870S; 1868, 2921;
1872, 1837

Seager, L. D., 1565, 1449

Seal, U. S., 1150, 1023

Searcy, R. L., 1034, 142

Searle, G. W., 541, 704

Seegers, W. H., 1151, 40

Seegmiller, C. G., 987, 924

Segal, S., 1152, 1064

Sehon, A. H., 542, 974

Seibert, R. A., 1531, 76; 1591, 1585

Seifter, J., 1291, 88; 1302, 1657; 1365, 1468;
1394, 1485; 1546, 1642; 1566, 87; 1668,
1759

Seki, S. L., 763, 928

FEDERATION PROCEEDINGS

Seletz, J. M., 1726, 1736

Selitto, J. J., 1567, 1623

Selkurt, E. E., 543, 674

Sellers, A. L., 1997, 449

Semple, R. E., 544, 572

Sendroy, J., Jr., 311, 579

Seraidarian, K., 1141, 1252

Serlin, I., 1725, 193

Setliff, J. A., 687, 861

Sevag, M. G., 911, 915; 1755, 916

Severinghaus, J. W., 545, 482; 590, 682

Sewell, G. E., 693, 829

Sexter, M. 8., 430, 226

Shadle, O. W., 58, 206

Shanes, A. M., 546, 252

Shank, R. E., 753, 1199

Shanor, S. P., 1289, 172; 1368, 1689; 1374,
176; 1568, 174

Shapira, R., 1154, 891

Shapiro, D. M., 547, 700

Shapiro, H., 548, 529

Shapiro, H. 8., 1153, 1051

Share, L., 549, 591

Sharma, G. P., 1668, 1759

Skaug, O., 1044, 1100

Shaw, K. N. F., 1155, 1171

Shaw, W. N., 1156, 1060

Shay, H., 1145, 973

Sheffner, A. L., 1157, 1900

Shelata, S., 698, 1076

Shelton, M., 1569, 1408

Shemano, I. 1570, 1577

Shemin, D., 1868S

Shellabarger, C. J., 1697, 139

Shepard, C. C., 1998, 1944

Shepard, R. H., 550, 303

Sheppard, H., 1352, 1436; 1571, 1529;
1603, 1576

Shepperd, I. M., 1369, 1427

Sherman, F., 402S

Sherman, F. G., 551, 78

Shermer, A., 668, 701

Sherrod, T. R., 1453, 1487

Sherwin, J. C., 1339, 107

Sherwood, W. W., 462, 262

Shettles, L. B., 552, 789

Shideman, F. E., 1582, 1385

Shigeura, H., 790, 948

Shimura, H., 332, 662

Shinaberger, J. H., 1572, 1488

Shinowara, G. Y., 1158, 37

Shkolnik, S., 1720, 1766

Shmukler, H. W., 1072, 1014

Shneour, E. A., 1159, 926

Shock, N. W., 274S; 448, 229; 556, 198

Shofer, R., 1577, 1440

Shohet, 8. S., 728, 1282

Shore, B., 1291, 88

Shore, P. A., 1356, 1636; 1415, 1638; 1423,
1631; 1465, 1630; 1561, 1643; 1573, 1629

Shorr, E., 19,; 576, 675; 1292, 1664

Shoulders, H. H., Jr., 553, 152

Shreeve, W. W., 1160, 1058

Shug, A. L., 1161, 1224

Shull, K. H., 670, 47; 1878, 1918

Shulman, §., 1290, 122; 1999, 2019

Shuster, L., 554, 1212

Sicé, J., 1726, 173€

Sidbury, J. B., Jr., 1162, 1094

Sideman, M. B., 1627, 1397

Sidlofsky, 8., 274, 536

Siebert, G., 1163, 1217

Siegel, H. 1876, 9

Siegmund, E. A., 1400, 1418; 1574, 1535

Volume ij

Siekevitz, P., 1164, 984

Sievers, M. L., 435, 479

Sigg, E. B., 1501, 1528; 1603, 1576

Silberberg, M., 1727, 1799

Silberberg, R., 1727, 1799

Silberberg, 8., 555, 2026

Silver, M. J., 616, 794

Silverman, M. 1165, 1328

Silverman, M. 8., 2000, 162

Silverstone, F. A., 556, 198

Silverstone, J. T., 49, 531

Sime, J. T., 1784, 1828; 1869, 1875

Simet, L., 1135, 820

Simmons, D. H., 557, 358

Simmons, E. L., 1728, 1750

Simmons, R. L., 659, 60

Simmons, V. P., 1575, MP, p. 628

Simms, E. 8., 950, 1185

Simonson, E., 558, 491

Simpson, M. E., 122, 521

Simpson, M. V., 1018, 881

Simpson, W. L., 1924, 1981

Singer, S., 807, 936

Singer, T. P., 932, 1233; 1011, 1234

Singleton, W. S., 708, 1347

Sinisterra, L., 1879, 1904

Sinsheimer, R. L., 1166, 1053

Sirny, R. J., 1167, 859

Sirota, J. H., 270, 349

Sisson, G. M., 1402, 1540; 1576, 1541

Sivertsen, L. N., 178, 752

Sizer, I. W., 921, 1030

Sjodin, R. A., 559, 250

Sjoerdsma, A., 1577, 1440

Skelton, F. R., 1729, 1788

Skipper, H. E., 710, 53

Slabok, M., 1438, 1653

Slade, H. D., 1902, 2055

Slanetz, C. A., 1691, 1899; 1692, 1%
1730, 1800

Slaton, W. H., Jr. 1023, 1293

Slaunwhite, W. R., Jr., 1168, 1164

Sloane, N. H., 1169, 1247

Sloboda, A. S., 1218, 98

Slocombe, A. G., 560, 322

Slocum, A C., 963, 939

Slovik, N., 1171, 1134

Slusher, M. A., 561, 364

Smadel, J. E., 1899, 1988; 1935, 2084

Small, W. J., 92, 664

Smart, J. O., 656, 420

Smith, A. H., 1851, 1856; 1866, 1879

Smith, A. H., Jr., 1599, 1575

Smith, C. C., 1578, 1446

Smith, C. M., 1579, 1433

Smith, D. C., 195, 515

Smith, Donald C., 1900, 1947

Smith, D. G., 1905, 1994; 2001, 1990;
1996

Smith, D. J., 1580, 1563

Smith, E. L., 361, 571

Smith, Emil L., 939, 1108

Smith, F., 562, 161

Smith, F. G., 897, 1368

Smith, F. H., 1402, 1540

Smith, G. M., 1401, 1496

Smith, J. D., 891, 1256

Smith, J. J., 563, 672

Smith, J. M., 1870, 1903

Smith, J. T., 1212, 1345

Smith, L., 1170, 1019

Smith, P. K., 1429, 1562 1464, 1551;
1550; 1478, 1684

Smith, R., 1952, 2028

ee a of oe Oe Se com ee ee, ek es es as es ccs ess ee a ce

‘olume i

875

». 628

6, 1544

1692, 1%)

, 1164

35, 2084

36, 1879

1
1, 1990; 20)

sss

March 1966

Smith, R. C:, 243, 285

Smith, R. E., 812, 1364

Smolens, J., 2002, 1962

Smothers, J. L., 610, 457

Smyrniotis, P. Z., 902, 947

Snell, K. C., 1734, 1743

Snider, R. S., 131, 266

Snyder, F. F., 564, 790

Snyderman, 8. E., 1871, 1861

Sobel, A. E., 1171, 1134

Sobell, S. D., 1766, 1849

Soderberg, T. E., 1770, 1857

Sokoloff, L., 565, 372; 1731, 1764

Solomon, A. K., 456, 617; 566, 619

Solomon, B. A., 1581, 1891

Solomon, C., 1826, 1894

Solomon, 8., 567, 223

Somers, L. M., 1318, 1434

Sommer, E., 1298, 1621

Sommers, 8. C., 1732, 1732

Sonnenschein, R. R., 568, 475

Sorof, S., 1172, 92

Sossen, R., 95, 111

Spackman, D. H., 1173, 1339

Spar, I. L., 569, 2003; 1645, 1758

Spearing, C. W., 863, 1308

Spector, E., 1582, 1385

Spector, H., 1157, 1900

Spector, S., 1583, 1511

Speirs, R. S., 163

Spencer, D., 1174, 856

Spencer, H., 570, 110; 1328, 1482

Spencer, M. P., 146, 337; 571, 433

Sperling, F., 1584, 1447

Sperry, R. W., 436, 777

Sperry, W. M., 1175, 853

Sphire, R. D., 1401, 1496

Spiegel, E. A., 572, 270; 1294, 1665

Spiegel-Adolf, M., 1176, 124

Spink, W. W., 398, 676; 1890, 2039

Spinzia, J., 515, 1494

Spirtes, M. A., 1585, 1510

Spitzer, J. J., 573, 86

Spitzer, J. R., 977, 1267

Spizizen, J., 1177, 1190

Spoerlein, M. T., 1586, 1617

Spoto, A. P., Jr., 1644, 1715

Sprague, J. M., 574, 791

Springer, G. F., 2003, 2047

Springs, V., 1182, 1158

Sprinson, D. B., 1179, 900

Sprinz, H., 2004, 2036

Sproul, E., 1672, 1728

Spruth, H. C., 1178, 1888

Spurr, G. B., 308, 764; 575, 677

Squibb, R. L., 1872, 1887

Srikantia, S. G., 19, 576, 675; 1292, 1664

Srinivasan, P. R., 1179, 900

Stacey, C. H., 800, 1773

Stacy, R. W., 481, 430

Stadie, W. C., 1156, 1060

Stadtman, E. R., 1180, 1085

Stahmann, M. A., 1181, 1958

Stainsby, W. N., 71, 381; 99, 380; 577, 288

Stamler, J., 578, 792; 579, 503

Stamler, F. W., 1733, 1741

Stanley, W. M., 869S

Stare, F. J., JS, p. 628; 1763, 8; 1790, 10;
1858, 7

Stark, L., 580, 471

Staron, N., 1445, 175

Starr, I., 581, 431

Stasilli, N. R., 367, 235

Stasney, J., 460, 408

err,

AUTHOR INDEX

Staub, A., 1182, 1153

Stavitsky, A. B., 2082S; 2005, 2007; 2031,
2006

Stavney, L., 1624, 1701

Steele, R., 8, 312; 637, 311; 720, 1386

Steelman, S., 1850, 1828

Steenbock, H., 783, 1280; 1779, 1927

Stefanini, M., 401, 116

Steffee, C. H., 1734, 1743

Steggerda, F. R., 582, 612

Steigmann, F., 1587, 1644; 1588, 1696

Stein, A. M., 1183, 1251

Stein, E., 525, 258

Stein, S. N., 339, 687; 468, 692; 490, 367

Stein, W. H., 874S; 1173, 1339

Steinberg, D., 1184, 884

Steinberg, G., 1023, 1293

Steinfeld, J. L., 583, 129

Stekol, J. A., 1185, 1249

Stelos, P., 2006, 1978

Stempfel, S. J., 1854, 1816

Stephanson, L., 236, 609; 1589, 1641

Stephenson, M. L., 1186, 877

Stephenson, N. R., 1187, 1349

Stern, H., 1188, 1103

Stern, J. R., 1189, 1082

Stern, K., 1908, 2046; 2007, 2045

Stern, K. G., 1116, 1129

Stetten, D., Jr., 815, 1054; 1190, 1314

Stetten, M. R., 1190, 1314

Stevens, C. M., 1191, 1307

Stevens, G. T., 451, 208

Stevens, K. M., 2008, 2005

Stevens, N., 961, 109

Stevens, V. L., 797, 1049

Stewart, B., 1888, 1821

Stewart, P. A., 584, 268

St. George, R. C. C., 904, 961

Stickney, J. C., 585, 705

Stiffey, A. V., 730, 1806

Stimmel, B. F., 1192, 1266

Stimpert, F. D., 2013, 1952

Stish, R. J., 398, 676

Stjernholm, R., 969, 1089

St. John, R., 439, 589

Stock, C. C., 859, 1034

Stoelting, V. K., 1407, 1465; 1333, 1464

Stohlman, F., Jr., 586, 212

Stokes, J., Jr., 2002, 1962

Stoll, A. M., 587, 728

Stone, C. A., 1590, 1429

Stone, D., 380, 315; 588, 1291; 723, 1158

Stone, H., 530, 499

Stotz, B., 1005, 1000; 1264, 999

Stracher, A., 1193, 1037

Strassman, M., 1194, 1308

Strates, B., 1195, 1133

Straube, R. L., 589, 182

Strauss, W. F., 658, 629

Strawitz, J. G., 1196, 989

Strawn, J. R., 1450, 1586; 1591, 1585

Strecker, H. J., 809, 1149

Strength, D. R., 1111, 1168

Strickland, K. P., 1020, 1004

Strong, J. P., 1735, 1

Struglia, L., 1823, 1881

Stumpe, M., 1737, 1776; 1741, 68

Stumpf, P. K., 852, 925; 1197, 932

Stupfel, M., 545, 482

Stupfel, M., 590, 682

Sturkie, P. D., 591, 560; 642, 798

Sturtevant, F. M., 1592, 1654

Suda, I., 592, 484

Sudsaneh, 8., 1807, 1914

Sullivan, J. G., 1601, 1530
Sullivan, W. J., 121, 253
Sunderman, F. W., 1209, 1479
Sure, B., 1873, 1928
Suskind, 8. R., 2009, 1985
Sutfin, D. C., 593, 1082
Sutherland, E. W., 1091, 1155
Sutherland, G. B., 594, 36
Sutherland, J. H. R., 595, 428
Sutin, J., 258, 416
Sutton, W. B., 1198, 1225
Svacha, R. L., 1099, 1885
Swain, H. H., 1593, 1491
Swan, H. J. C., 352, 569
Swann, H. G., 596, 222
Swanson, H. E., 597, 538
Swanson, M., 1199, 1009
Swanson, P., 1769, 1909; 1793, 1845; 1874,
1908; 1889, 1854
Sweat, M. L., 1200, 1159
Sweet, N. J., 171, 708
Swendseid, M. E., 1875, 1841
Swenson, E. M., 1539 ,1463
Swern, D., 1692, 1797
Swick, R. W., 945, 1181; 1736, 1254
Swift, A., 1942, 2004
Swift, M. N., 599, 31
Swim, H. E., 1926, 1938
Swinehart, L. A., 1719, 1708
Swingle, W. W., 30, 517
Swintosky, J. V., 1375, 1553
Sydenstricker, V. P., 1802, 1842
Symochowicz, S., 240, 464
Syverton, J. T., 1919, 1969
Szekely, E. G., 572, 270; 1294, 1665

TV abechaik, I. I. A., 1594, 1542

Tabachnick, J., 1749, 141; 2010, 826

Tabern, D. L., 962, 1320

Tabor, C. W., 1201, 865; 1595, 1616A

Tabor, H., 1201, 865

Tahmisian, T. N., 598, 69

Takagi, Y., 1202, 1124

Takahashi, H., 1174, 856

Takemoto, K. K., 2011, 1945

Takenaka, Y., 1148, 1098

Taketa, S. T., 599, 31

Talalay, P., 1203, 1163

Talbert, P. T., 1204, 1079

Taliaferro, W. H., 1936, 159; 2012, 1977

Tallan, H. H., 1205, 1138

Talmage, D. W., 2006, 1978; 2012, 1977

Talmage, R. V., 83, 414

Tammes, A. R., 796, 1335

Tamura, R., 1737, 1776

Tandon, O. B., 1868, 1921

Tang, P. C., 600, 301

Tannenbaum, M., 601, 793

Tanz, R. D., 1596, 1409

Tarleton, G. J., 1921, 1794

Tarr, H. L. A., 1206, 1121

Tauber, H., 1207, 1713

Taylor, A., 602, 519

Taylor, A. R., 2013, 1952

Taylor, C. B., 1737, 1776; 1741, 68

Taylor, H. L., 255, 500; 428, 505a; 603,
505b; 1762, 3

Taylor, J. D., 1539, 1463; 1597, 1537

Taylor, W. R., 1208, 898

Taylor, Z., 99, 380; 586, 212

Teasdall, R. D., 604, 468
Tedschi, R. E., 1209, 1479
Tein. A. B., 922, 41

XViii

Telford, J., 1447, 1502

Telka, M., 1244, 890

Tennent, D. M., 1876, 9

Teply, L. J., 182S

Terayama, H., 1227, 832

Terminiello, L., 1210, 887

Terner, C., 605, 1080

Terzin, A. L., 2014, 1989

Terzuolo, C. A., 606, 244

Thal, A. P., 370, 565

Thannhauser, 8. J., 717, 1344; 1141, 1252

Thayer, P. S., 1598, 1544

Thayer, S. A., 903, 852

Theis, G., 1946

Thiers, R. E., 1211, 978

Thiessen, C. P., 712, 1218

Thomas, A. J., 1194, 1303

Thomas, G., 1438, 1653

Thomas, L., 2015, 2050

Thomas, L. E., 1212, 1345

Thompson, D. D., 607, 297

Thompson, W. D., 1263, 967

Thomson, J. F., 233, 618

Thorne, C. B., 170, 1105

Thurlow, J., 1106, 1203

Thygeson, P., 1937, 2017

Tidwell, H. C., 1771, 1846

Tietz, A., 1234, 1286

Tildon, J. T., 1846, 1874

Tillotson, C., 608, 409

Timiras, P. S., 609, 525; 1631, 1426

Timm, E. A., 1967, 1948; 2013, 1952; 2016,
1951

Tipton, C., 1063, 952

Tipton, S. R., 4038S; 610, 457

Tislow, R., 1365, 1468; 1432, 1656

Titus, D. C., 1625, 1038

Toal, J. N., 1751, 32

Tobias, G. J., 488, 345

Tocantins, L. M., 98, 153; 1537, 1691

Tokizane, T., 176, 750

Tolbert, N. E., 1213, 944

Tollin, G., 1214, 892

Tolmach, L. J., 2017, 2061

Toman, J. E. P., 1369, 1427; 1599, 1575;
1600, 1697

Tomashefsky, P., 824, 56; 838, 1365

Tomich, E. G., 1415, 1638; 1465, 1630

Tomizawa, H. H., 1738, 1154

Toolan, H. W., 1739, 1703

Toporek, M., 1740, 46

Topper, Y. J., 1215, 1096

Torchiana, M. L., 1590, 1429

Torre, A. V., 400, 707

Torriani, A. M., 1130, 1206

Totter, J. R., 778, 1241

Touchstone, J. C., 269, 318; 611, 592

Touster, O., 1216, 945

Towne, J. C., 1217, 1215

Townsend, P., 1546, 1642

Townsley, P. M., 1045, 1312

Toyama, S., 109, 493

Trafton, H. M., 1306, 1554

Trapani, I. L., 192, 576

Trapani, R., 1916, 2035

Trapold, J. H., 1601, 1530

Trautman, R., 1237, 976

Travis, H. F., 1877, 1807

Treat, A. E., 612, 598

Tredwell, C., 1819, 1922

Trémége, M., 919, 1310

Trew, J. A., 703, 50

Trousof, N., 1482, 1389

Troy, W. P., 1218, 98

FEDERATION PROCEEDINGS

Trueheart, R., 1741, 68
Truitt, E. B., Jr., 1602, 1514
Trujillo, T. T., 1219, 1337
Trulson, M. F., 1883, 1916
Trunnell, J. B., 791, 1248
Tseng, K. C., 1033, 918
Tsien, W. H., 1603, 1576
Ts’o, P. O. P., 1220, 1102
Tsuyuki, H., 1181, 1958
Tucker, B. E., 299, 374
Tucker, D., 43, 605; 613, 604
Tunik, B. D., 614, 256
Tureman, J. R., 1604, 187
Turino, G. M., 615, 355
Turner, D. L., 616, 794
Turner, M. D., 617, 319
Turner, M. L., 1101, 55
Tuttle, W. W., 520, 507
Twombly, G. H., 977, 1267
Tyberghein, J. M., 1738, 1154
Tyler, D. B., 618, 903

Usentriena, S., 972, 1156; 1311, 13;
1356, 1636; 1465, 1630; 1605, 1635

Uhr, J. W., 2018, 2028

Ulin, A. W., 251, 45

Umbarger, H. E., 1221, 1305

Umberger, E. J., 1222, 1649

Ungar, G., 619, 657

Unglaub, W. G., 1223, 1920; 1799, 1814

Unna, K. R., 1579, 1433

Upham, H. C., 1358, 1615

Upton, A. C., 1663, 1793; 1742, 1748

Utter, M. F., 1051, 1017

Uyeki, E. M., 1606, 75

Vikas: J. S., 1854, 1816

Valk, A. deT., Jr., 1525, 1557

Vallee, B. L., 300, 835; i211, 978; 1639,
831; 2019, 1099

Valles, W. C., 494, 616

Vanamee, P., 620, 497

Van Arman, C. G., 1443, 1619

Vanatta, J. C., 621, 393

Vanderhoff, G. A., 992, 622

Vander Wende, C., 1607, 1499

van Eys, J., 1224, 834

Van Fossan, D. D., 112, 584; 622, 586;
650, 585

Vangerov, M., 735, 1193

van Hees, G. R., 1289, 172; 1374, 176;
1568, 174

Van Itallie, T. B., 1878, 1918

Van Liere, E. J., 585, 705; 623, 795

Van Liew, H. D., 624, 582

Van Maanen, E. F., 1463, 1520

Van Middlesworth, L., 317, 478; 625, 796

Van Reen, R., 700, 1021

Van Vunakis, H., 732, 1112; 2020, 1186

van Wagtendonk, W. J., 1274, 937

Van Winkle, 8., 1304, 1625

Vars, H..M., 1225, 1780

Vaughan, B. E., 347, 770

Vaughan, J. H., 2021, 1966

Vaughan, L., 1792, 1902

Vaughan, M., 1184, 884

Vaughn, C., 1007, 1220

Velardo, J. T., 626, 797

Velasquez, T., 479, 583

Veldhuis, A. H., 1719, 1708

Vennesland, B., 978, 818

Volume 16

Venning, E. H., 800, 1773
Vergeer, T., 172, 630

Vernier, V. G., 627, 597
Vernon, L. P., 1226, 1239
Vesely, B. M., 951, 855
Vestling, C. S., 1227, 882

Vick, R. L., 1608, 1411

Villaca, L., 243, 285

Villee, C. A., 1228, 1290
Vinograd, J., 1220, 1102
Vishniac, H. S., 2022, 849
Visscher, M. B., 279S; 379, 394; 398, 676
Visser, D. W., 1979, 1995

Vitale, J. J., 1677, 1788; 1879, 1904
Vogel, F. S., 229, 105

Vogel, H. H., Jr., 628, 28

Vogel, H. J., 1131, 866

Vohra, P., 1191, 1307; 1822, 1859
Volk, B. W., 1748, 114

Volk, W. A., 1229, 1316

Volkin, E., 1230, 1192

Volwiler, W., 248, 807

Von Euler, C., 176, 750; 629, 601
von Haam, E., 1744, 1754

von Hippel, P. H., 630, 803
von Kaulla K. N., 631, 156
Von Korff, R. W., 1231, 1081
Vo-Vinh-Hoa, 126, 742

Wabnitz, C. H., 1015, 1373
Wachsman, J. T., 697, 941
Wachstein, M., 1745, 1761
Waddell, W. J., 1609, 627
Wade, O. L., 120, 451; 645, 330
Wadkins, C. L., 1232, 1237
Wager, O. A., 2029S

Wagle, G., 1284, 1579
Wagman, I. H., 632, 535
Wagner, C., 898, 943

Wagner, J., 1594, 1542
Wagner, M. L., 1138, 970
Wagner, R. H., 1746, 113
Wainio, W. W., 1233, 1230
Wakerlin, G. E., 133, 743; 208, 564
Wakil, S. J., 1234, 1286
Wakim, K. G., 633, 551
Waksman, B. H., 2023, 2049
Walaszek, E., 634, 373
Walaszek, E. J., 1610, 1438
Wald, G., 1235, 962

Waldorf, M. A., 1132, 1919
Waldow, A., 243, 285
Waldron, J. M., 187, 660
Walker, B. S., 983, 1123
Walker, J. B., 1236, 1300
Walker, M., 1922, 131

Walker, R. M., 568, 475
Walker, S. M., 635, 492
Walker, W. G., 245, 438; 636, 574
Wall, J. S., 8, 312; 637, 311
Wall, P. D., 328, 470

Wall, R. L., 458, 237
Wallenius, G., 1237, 976
Wallenstein, S. L., 1434, 1501; 1611, 1506
Walser, M., 1747, 1765

Walter, H., 880, 810

Walton, R. P., 1351, 289
Wander, H. J., 75, 224

Wang, C. H., 1238, 1379
Wang, G. H., 638, 466

Wang, S. C., 265, 271; 1431, 1570
Wanner, R. L., 1880, 1925
Ward, J. W., 1612, 1612

Mar

Ware
Warn
Warn
Warn
Warn
Warn
Warn
Warre
Wars!
Warts
Wase,

Wass
Watje
Watk
Watk
Watsc
Watsc
Watsc
Watts
Watts
Waud
Waug
Wax,

Way,

Weatl
Weave
Webst
Wedg
Weigh
Weike
Weil, .
Weil,

Weil,
Weil,
Weim!
Weim
Weine
Weinh
Weink
Weinn
Weins
Weins
Weint:

Weise,

me 16

, 1506

March 1956

fare, F., 1491, 1519; 1613, 1698
Warner, G. F., 160, 333

Warner, N., 1748, 1781

Warner, R. C., 979, 969; 1239, 1257
Warner, R. G., 1877, 1807

Warner, W. D., 1862, 1805
Warnock, N. H., 1456, 1441
Warren, L., 1240, 912

Warshaw, L., 1404, 1555

Wartman, W. B., 1689, 1740

Wase, A. W., 1241, 1819; 1336, 1437; 1614,
1699

Wasserman, R. H., 1881, 1865
Watjen, A., 1185, 1249

Watkins, D. H., 1553, 1448

Watkins, L. C., 1701, 1762

Watson, H., 1449, 626

Watson, M. L., 1164, 984

Watson, M. S., 2024, 1996

Watts, D. T., 1615, 1560

Watts, L. E., 1472, 1425

Waud, R. A., 1616, 1616

Waugh, D. F., 630, 803; 666, 1342

Wax, D., 1617, 1450

Way, E. L., 1391, 1588

Weatherby, J. H., 1493, 1390

Weaver, R. H., 1242, 867

Webster, G. C., 1243, 876

Wedgwood, R. J., 2025, 2069

Weigle, W. O., 2031S; 2026, 2065

Weikel, J. H., Jr., 1618, 1398

Weil, J., 631, 156

Weil, L., 1244, 890

Weil, M. H., 398, 676

Weil, W., 515, 1494

Weimberg, R., 794, 950

Weimer, H. E., 2027, 2038

Weiner, I. M., 639, 342

Weinhouse, S., 1194, 1303

Weinke, K. F., 1181, 1958

Weinman, E. O., 1245, 1380

Weinstein, V. A., 304, 615

Weinstock, I. M., 1882, 1876

Weintraub, R. L., 1046, 930

Weisberg, J., 1448, 1513

Weise, V. K., 413, 518

Weiser, R. S., 2028, 1943

Weiss, A. K., 640, 542

Weiss, B., 641, 1002

Weiss, C., 1749, 141

Weiss, H. S., 591, 560; 642, 798

Weiss, S., 1185, 1249

Weiss, S. B., 1246, 1003

Weissbach, H., 1311, 1634; 1605, 1635

Weissmann, B., 1247, 919

Weisz, A., 529, 788

Welch, A. D., 871, 96

Weich, G. H., 59, 210; 71, 381; 577, 288

Weller, J. M., 242, 186

Wellington, F. M., 1228, 1290

Wells, I. C., 1248, 1067

Wells, J. A., 1405, 1489; 1451, 1469

Wells, J. G., 21, 512; 179, 681; 643, 680

Wells, W., 1249, 48

Wendel, H., 1466, 1504; 1619, 1503

Werbin, H., 1250, 1265

Werder, A. A., 1750, 1771

Wertz, A. W., 1827, 1932

West, C. D., 108, 397

West, E. S., 37, 709

West, F. R., 1620, 1613

West, J. W., 644, 384

West, T. C., 513, 281; 1621, 1454

Westerfeld, W. W., 895, 1883

AUTHOR INDEX

Westfall, B. A., 1622, 1700
Westheimer, F. H., 675, 815
Weston, J. K., 237, 1478; 1489, 1406
Weston, R. E., 318, 459
Westphal, U., 1251, 1358
Westrick, M. L., 1678, 1768
Weymouth, P. P., 1252, 72
Wharton, F. D., Jr., 1794, 1839
Wheaton, R. R., 1499, 1417
Wheeler, H. O., 120, 451; 645, 330
Whitaker, J. R., 1253, 819

White, A., 993S

White, A. G., 646, 291

White, C. W., Jr., 1495, 1500
White, J., 1751, 32

White, J. L., 708, 1347

White, J. M., 1623, 1624

White, J. M., Jr., 1624, 1701
White, P. L., 1879, 1904; 1883, 1916
White, R. P., 647, 269
Whitehorn, W. V., 290, 683
Whiteside, J. E., 1756, 2018
Whittaker, V. P., 1353, 173
Wick, A. N., 1254, 1065
Wiebelhaus, V. D., 1625, 1038
Wiercinski, F. J., 648, 254

Wiese, H. F., 1884, 1852
Wiggans, D. S., 1255, 808

Wight, R., 1864, 1936

Wikler, A., 185, 379; 1377, 1505
Wilber, C. G., 649, 799

Wilcox, P. E., 1256, 1111
Wilgram, G. F., 1257, 1742
Wilhelmi, A. E., 901, 1791
Wilkie, M. H., 2029, 2044

Wilkin, D. R., 873, 843

Wilkins, C. N., Jr., 1971, 917
Wilks, J. W., 1632, 1492

Wilks, S. S., 112, 584; 622, 586; 650, 585
Williams, A. D., 508, 785
Williams, F. L., 342, 759
Williams, H. H., 1829, 1917
Williams, H. L., 584, 268
Williams, J., 114, 272

Williams, J. K., 651, 221
Williams, J. N., Jr., 1259, 1381; 1774, 1815
Williams, M. W., 652, 800
Williams, R. B., 339, 687
Williams, R. B., Jr., 1751, 32
Williams, R. H., 1738, 1154
Williams, T. L., 94, 1260
Williams, W. L., 653, 801; 808, 1785
Williams-Ashman, H. G., 1258, 897
Williamson, B. J., 654, 498
Williamson, M. B., 1260, 1382
Willman, V. L., 125, 650

Wills, J. H., 1626, 188

Wilson, H., 811, 1165

Wilson, M. A., 697, 941

Wilson, P. W., 1161, 1224
Wilson, R. H., 1627, 1397
Wilson, T. H., 1261, 899

Wilson, V. J., 655, 474

Wiltshire, L. L., 1339, 107
Winbury, M. M., 1526, 1601; 1628, 1419
Windle, W. F., 656, 420
Windmueller, H. G., 1262, 1893
Wingo, W. J., 1263, 967

Winitz, M., 718, 1808; 1214, 892
Winnick, T. C., 593, 1032
Winter, K. K., 1752, 1746
Winter, W. I., 209, 335
Winternitz, W. W., 657, 710
Wintrobe, M. M., 1801, 1887

Winzler, R. J., 1249, 48; 1720, 1766
Wirts, C. W., 64, 737

Wiseman, R., 1437, 1640
Wislicki, L., 1629, 1521

Wissler, R. W., 1706, 1781; 1753, 12
Witebsky, E., 1987, 2020

Witt, D. M., 346, 24

Witter, R. F., 1005, 1000; 1264, 999
Wittler, R. G., 2030, 2060
Wixom, R. L., 1885, 1895
Wizerkaniuk, M., 784, 1066
Woebke, J., 1893, 2052

Woessner, J. F., Jr., 694, 1068
Wolbach, R. A., 658, 629
Wolbarsht, M., 261, 320

Wolf, A. V., 511, 786

Wolf, B., 2031, 2006

Wolf, D. E., 1852, 1890

Wolf, G., 883, 1302; 965, 1279
Wolf, L. M., 155, 746; 237, 1478
Wolf, M. M., 1630, 1486

Wolfe, D. E., 569, 2003

Wolf, J. B., 438, 901

Wolff, E. C., 1265, 1324

Wolff, H. G., 50, 734; 1515, 1424
Wolff, J., 1265, 1324

Wolpert, A., 1602, 1514

Wong, H. Y. C., 659, 60

Wong, J., 930, 1057

Wood, D. C., 1754, 1723

Wood, E. H., 207, 570; 352, 569
Wood, H. G., 994S; 1266, 951
Wood, J. E., 2032, 631

Wood, R. C., 1755, 916

Woodard, G., 1414, 1605
Woodbury, D. M., 1458, 1539; 1631, 1426
Woodbury, J. W., 70, 395
Woodbury, R. A., 1632, 1492
Woodcock, A. H., 660, 633
Woodruff, C. W., 1886, 1937
Woods, E. F., 1540, 1558

Woods, L. A., 1633, 1444

Woods, M., 1267, 99

Woodson, C. S., 822, 169
Woodward, H., Jr., 132, 196
Woodworth, P., 181S

Woolley, D. W., 1028, 1107
Woolridge, R. L., 1756, 2018
Wrenshall, G. A., 102, 523; 602, 519
Wright, B. E., 1268, 1331
Wright, B. J., 598, 69

Wright, E. B., 661, 247

Wright, H. V., 1787, 1817; 1844, 1827
Wright, J. H., Jr., 1058, 1062
Wright, J. L., 662, 283

Wright, N. G., 721, 1144
Wunder, C. G., 663, 648

Wyatt, G. R., 1269, 1311

Wycis, H. T., 1176, 124

Wycoff, H. D., 1270, 115
Wyngaarden, J. B., 1152, 1064; 1271, 920
Wyss, O., 1272, 1383

pees ieg R. S., 1887, 1892
Yamanouchi, I., 434, 889
Yamasaki, S., 224, 375
Yanari, S. S., 1970, 1340
Yang, C. S., 1888, 1821
Yang, S. P., 1889, 1854
Yarbro, C. L., 1273, 1384
Yard, A. C., 1634, 1597
Yates, F. E., 24, 502

“XX

Yenson, M., 1980, 2067
Yiengst, M. J., 556, 198
Yim, G. K. W., 1635, 1598
Yohe, M., 1792, 1902
Yonce, L. R., 664, 192
Yoneda, M., 2033, 2048
Yoo, A. E. J., 1636, 1549
Young, A. C., 600, 301; 665, 354
Young, D. E., 1869, 1875
Young, E. M., 1172, 92
Young, G. R., 1274, 937
Young, J. V., 1701, 1762
Young, P., 1491, 1519
Young, W. P., 1048, 844
Youngner, J. S., 2034, 1949
Yphantis, D. A., 666, 1342

FEDERATION PROCEEDINGS

Yi, T. F., 36, 218; 270, 349
Yung, N. C., 828, 840
Yurochko, F., 1992, 1950

Zain, 1., 1159, 926
Zakrzewski, S. F., 1275, 1333
Zamcheck, N., 1677, 1788; 1879, 1904
Zamecnick, P. C., 1186, 877
Zamenhof, S., 1276, 1183
Zannoni, V. G., 1277, 1170
Zaratzian, V. L., 1416, 1609
Zarrow, M. X., 667, 411
Zuder, H. L., 1637, 1610
Zavattaro, D. N., 680, 1284
Zawoiski, E. J., 668, 701

Volume tj

Zeitinger, J. R., 1988, 2056

Zeller, E. A., 1278, 888

Zemel, R., 102, 523

Zeigler, D. M., 974, 1016

Zierler, K. L., 13, 549; 245, 438; 282, 554
Zill, L. P., 1279, 1318

Zillig, W., 1638, 1648

Zilversmit, D. B., 669, 57
Zimmerman, Sister M. A., 793, 854
Zittle, C. A., 1281, 1101

Zomzely, C., 670, 47

Zorn, E. M., 941, 837

Zubrod, C. G., 1381, 1394

Zuidema, G. D., 113, 505; 671, 504
Zweifach, B. W., 672, 802; 673, 673
Zwickey, R. E., 1380, 1403; 1757, 1783

‘olume i

8; 282, 554

93, 854

SUBJECT INDEX

Norte: This subject index is a modification of the system of coordinate
indexing (see Coordinate Indexing: M. Taube and Associates, Documenta-
tion Incorporated, 1953) using unit terms rather than cross references.

The user, rather than the indexer, determines what terms he wishes
to coordinate. By looking up each of these and noting the serial numbers
that are common, he can find the abstracts that combine the items. The
index may be used in the usual way for single item search. Index references
are the serial numbers of these abstracts.

Due to the short time available for preparation of the index, many com-
promises in unit terms have been necessary, and frequently general rather
than specific entries have been used.

Absorbie acid, 1859, 1860

Absorption, 213, 357, 389, 427, 582, 1208,
1643, 1809, 1850, 1851, 1853, 1881
Acclimatization, 21, 105, 150, 274, 294,
$29, 339, 347, 594

‘Acerola’ juice, 782

Acetal phosphatides, 1068

Acetate, 194, 861, 930, 1034, 1160
Acetazoleamide, 540, 1458

Acetic acid, 1424

Acetoacetate, 772, 1231

Acetoacetyl glutathione thioesterase,
1189

Acetobacter suboxydans, 943

Acetylable steroids, 714
N-acetyl-L-aspartic acid, 1205

Acetylcholine, 14, 47, 1372, 1408, 1579

Acetylcholinesterase, 1408, 1725

Acetyl digitoxin, 1617
N-acetylglucosamines, 919

Acetyl strophanthidin, 515
Acetylthiocholine, 1021
Acetyltryptophan, 374

Acid aminopolysaccharides, 1102 «}

Acid-base, 179, 242, 303, 355

Acid secretion, 887

Acidosis, 74, 216, 488, 1845

Acidosis, diabetic, 815

Aconitase, 712

Acquired hemolytic anemia, 1118

ACTH, 30, 268, 417, 474, 526, 537, 670,
881, 1656

Action potential, 84

Active transport, 148, 245, 475, 536

Actomyosin, 614, 848

Acylase, 1037

deyl CoA dehydrogenases, 706

Acyl glucuronides, 1560

Adaptation, 110

Addiction, 185, 1377, 1378

Adenine, 909

Adenine nucleotides, 1076

Adenofibrosis, 1732

Adenosine, 695
Aa, * diph phate-ribose, 873

Adenosine-5’-phosphate, 1060

Adenosine phosphoryl carbonate, 694

Adenosine triphosphate, 63, 68

S§adenosylmethionine, 746

Adenyl-acylates, 713

Adenylic-5’-phosphoric acid deaminase,
971

Adenylosuccinate, 982

Adrenal glands, 53, 55, 190, 191, 195, 222,
234, 247, 277, 310, 323, 356, 410, 442, 459,
467, 480, 507, 537, 652, 657, 801, 804,
844, 881, 1422, 1443, 1469, 1485, 1487,
1656, 1658, 1685, 1690, 1710, 1729, 1770,
1839

Adsorption, 1746

Aeration, 1094

Age, 422, 448, 556, 558, 642

Agglutination, 1945, 2021

Aircraft accidents, 650

8-Alanine, 995, 1180

Albumin, 211

Alcohol, 1457

Alcoholchloral hemi-acetals, 1365

Alcohol dehydrogenase, 999, 1224

Aldosterone, 800

Alkali metal, 1104

Allergen, 1956

Alligator, 291, 769

t-Allothreonine, 926

Alloxan, 838, 1342, 1492

N-allylnormorphine, 1288, 1619

Alpha ketol, 912

Altitude, 7, 21, 110, 195, 347, 441, 479, 609

Amabacides, 1435

Amethopterin, 1381

Amino acids, 402, 736, 900, 914, 1016,
1018, 1082, 1085, 1104, 1119, 1126, 1167,
1178, 1186, 1248, 1382, 1519, 1770, 1782,
1785, 1803, 1843, 1851, 1855, 1881, 1888,
1889, 1926

Amino acid esterases, 1484

Amino acid-nucleotide complexes, 1075

p-Amino acid oxidase, 944, 1263

Aminobutyric acid, 894

Amino groups, 1256

5-Amino-4-imidazolecarb

5-Amino-4-imidazolecarb id
tide, 993, 1240

5 - Amino - 4 - imidazole - (N - succinylo-
carboxamide) ribotide, 998

6-Aminonicotinamide, 924

Amino nitrogen levels, 680

Aminopeptidases, 1061

Aminophylline, 220

8-Aminopropionitrile, 1661, 1705

Aminopterin, 1677, 1784

2-Amino-1,3,4-thiadiazole, 1218

Ammonia, 863, 1493

Amniotic fluid, 552

ide, 911
ribo-

xxi

AMP deaminase, 1027

Amphenone ‘B’, 507

Amphibia, 786, 833

Amylase, 20, 1693

Analgesics, 1304, 1331, 1396, 1401, 1448,
1567, 1607, 1623

Anamnestic response, 2027

Anaphylaxis, 1586

Androblastoma, 1748

5-Androstene-3, 17-dione, 1203

Androsti glucur: ide, 980

Anemia, 87, 128, 392, 411, 1654, 1781,
1801, 1875

Anesthetics, 96, 804, 1289, 1421, 1624

Aneurysm, 1734

Angiotonin, 737, 889

Anhydrocitrovorum factor, 1165

Anserine, 593, 1019

Antabuse, 1451

Anthelmintics, 1350

Anthranilic, 733

Anthrone, 1279

Anthropometry, 41,

Antibiotics, 816, 1953

Antibodies, 562, 569, 1118, 1653, 1703,
1713, 1715, 1754, 1756, 1859, 1896, 1911,
1912, 1913, 1941, 1951, 1954, 1963, 1974,
1984, 1987, 2002, 2005, 2008, 2012, 2018,
2029, 2031

Anticholinergic drugs, 1551

Anticholinesterase, 1403, 1504, 1547, 1604,
1626 A

Anticoagulants, 338

Anticonvulsants, 1296, 1402, 1574, 1594,
1597, 1589

Antidiuresis. 219, 530

Antifibrinolysin, 1029

Antigen, 1181, 1911, 1912, 1917, 1953,
1955, 1956, 1974, 2029

Antihistamines, 111, 1628

Antirabies vaccination, 1914

Antisera, 885, 1925

Antitoxins, 1948

Aorta, 563, 662, 1734, 1735

APC viruses, 1756, 1937, 1972

p-Arabinose, 794

t-Arabinose, 516, 1229

Aramine, 432

Arginase, 747, 837

Arginine, 837, 1825, 1861

Armadillos, 83

Arsanilic acid, 1178

XXli

Arsenite, 1199

Arteriosclerosis, 273,

Arteritis, 1676

Arterenol, 1490

Arthropod-borne virus diseases,

Arthus reaction, 1940

Artificial kidney, 1701

Ascaris, 1059, 1994

Ascites, 22, 876, 910, 986, 1061, 1714, 1901,
1922, 1946

Ascorbic acid, 18, 753, 782, 801, 987, 1054,
1106, 1122

Ash, 1840,

Asparagine, 1016, 1133, 1594

Aspartic acid, 1039, 1133

Assimilatory quotient, 739

Atherogenesis, 1876

Atheromatosis, 669,

Atherosclerosis, 165, 579, 659, 669, 704,
1469, 1735, 1763, 1790, 1831, 1858

ATP, 892, 1027, 1043

Atrophy, 279

Atropine, 1386, 1494, 1579, 1612, 1620

Aureomycin, 1842, 1868, 1875

Autolysis, 1260

Automatic recording apparatus, 1173

Autoperfusion, 1092

Autoprothrombin I, 1151

Auxins, 1379

Avocado, 676

Axone discharge, 60

Azacyclonol, 1592

8-Azaguanine, 807, 1002

Azaserine, 774

Azetazoleamide, 1406

Azide, 315, 861, 1199, 1552

Azo dyes, 1598

Azoproteins, 1172

Azotobacter, 861, 1272

6-Azuracil, 871

1510, 1709, 1741

1996

icsendas, 1670

Bacteria, 672, 1670, 1684, 1712, 1721, 1754,
1755, 1890

Bacteriophage, 692, 952, 1053, 1230, 2017,
2020

Bacterium tularense, 1232

Barbiturate, 96, 185, 225, 1377, 1407, 1442,
1482

Behavior, 14

Benadryl, 1921

Benzimidazoles, 674, 759

Benzoate, 944

Benzoyldipeptides, 1214

Besnoitia jellisoni, 1673

Betaine, 1067

Bicarbonate, 654

Bicycloheptenes, 1326, 1574

Bile, 541, 1115, 1225, 1647, 1707

Bile acids, 793, 830, 903, 1991

Bile duct. 1225, 1688

Biotin, 1971

Biphenyl, 1313

Blood, 71, 72, 211, 249, 369, 384, 504, 542,
601, 1449, 1609, 1855, 1872

Blood clotting, 64, 154, 278, 287, 322, 338,
358, 401, 421, 429, 493, 553, 594, 616, 908,
1642, 1702, 1743, 1757

Blood flow, 38, 93, 146, 160, 172, 272, 295,
1572

Blood glucose, 1796

FEDERATION PROCEEDINGS

Blood groups, 1931, 2003

Blood vessels, 307, 378

Blood volume, 136, 160, 207, 214, 361, 407,
544, 583, 636, 645

Body weight, 41

Bone, 186, 960, 1661, 1686, 1731, 1848,
1865

Bone marrow, 53, 419, 601

Brain, 14, 49, 60, 114, 188, 224, 225, 229,
261, 292, 299, 353, 366, 387, 388, 403, 438,
436, 461, 463, 476, 489, 514, 565, 568, 572,
627, 634, 968, 973, 1020, 1057, 1150, 1175,
1205, 1631

Bread, 1808, 1817

5-Bromouracil, 1166, 1276

Bufotenine, 1370

Burns, 139, 232, 259, 508, 587, 1747

Butter, 1856

Butylalcohol, 257

Butylated hydroxyanisole, 1794

-Butyrobetaine, 1816

Butyryl adenylate, 1204

Gitsiien 115, 780, 781

Caffeine, 768, 1321

Calcification, 1171, 1779

Calcium, 1043, 1272, 1491, 1798, 1819, 1865

Calcium; Ca*s, 224, 729, 1880, 1881

Caloric intake, 1788

Calories, 1862, 1879

Calorimeter, 45, 425

Camel, 532

Canavanine flavianate, 1464

Canine distemper, 1954

Capillaries, 4, 294

Capillary permeability, 417

Capillary resistance, 42

Carbamidines, 1483

Carbamyl phosphate, 773, 869

Carbohydrate, 8, 13, 48, 132, 203, 603,
657, 1421, 1730

Carbohydrate metabolism, 217, 296, 355,
446, 551, 910

Carbohydrate oxidation, 879

Carbohydrate synthesis, 1059

Carbohydrate tests, 975

Carbon; C™, 221, 720, 1656

Carbon dioxide, 339, 347, 375, 412, 447,
490, 527, 590, 1213, 1540

Carbonic anhydrase, 527, 620, 769, 981

Carbonic anhydrase inhibitors, 1402,
1576

Carbon monoxide, 615

C"-carboxyl-labeled nicotinamide, 1775

Carcinogen, 1744

Cardiac arrhythmias, 1364, 1471, 1477,
1553, 1565, 1613

Cardiac output, 71, 86, 232, 306, 352, 581

Cardiometer, 44

Carnitine, 835, 1816

Carnosine, 593

Carotene, 688

Carotenoids, 1159

Carotid sinus, 133, 286

Casein, 1262, 1854, 1861, 1904

Catalase, 1277

Cataracts, 1826

Catecholamines, 1367

Cathartic agents, 1404

Cations, 1025

Caudate nucleus, 131

C-C hydroylsis, 1154

Volume 15

Cell division, 239, 1674

Cellobiose, 1815

Cellulolytic bacterium, 1815

Central nervous system, 343, 592

Cephalins, 870

Cerebellum, 131, 182, 433, 584

Cerebral circulation, 198, 1472

Cerebral lesions, 50

Cerebroside, 1089

Cervix, 1744

Cesium, 1841

Chelates, 1541, 1542

Chenodeoxycholic, 1000

Chinook salmon, 1782

9 - (2 - Chlorethyl) ethylaminomethyl,
1498

Chloride, 129, 171, 245, 887, 1691

Chlorin derivative, 1441

Chlorisondamine, 1571

Chloroform, 1520

Chlorophyll, 1420

Chlorpromazine, 89, 446, 575, 656, 1294,
1336, 1337, 1341, 1355, 1368, 1419, 1440,
1454, 1460, 1467, 1486, 1496, 1508, 1524,
1554, 1592, 1614

Cholangiofibrosis, 1778

38-Cholestanol, 1647

Chlortetracycline, 1649

Cholesterol, 97, 213, 332, 385, 389, 395,
760, 829, 830, 862, 1056, 1065, 1114, 1707,
1737, 1759, 1762, 1790, 1831, 1849, 1856,
1858, 1876

Cholesterol esterase, 951

Choline, 156, 690, 1185, 1246, 1257, 1536,
1593, 1778, 1887

Cholinergic agents, 899

Cholinesterase, 242, 331, 664, 822, 937,
1021, 1289, 1322, 1353, 1368, 1374, 1445,
1533, 1568

Chondroitin sulfate, 753, 1012, 1139

Chromatography, 641, 736, 1005, 1039,
1173, 1970

Chylomicra, 853, 1771

a-Chymotrypsin, 866, 1135, 1253, 1425,
1646

Chymotrypsinogen, 1256

Circulation, 16, 22, 142, 201, 209, 271, 407,
506, 548, 568, 571

Cirrhosis, 1716, 1886, 1904

Citrate, 783, 856, 1273

Citrovorum factor, 681, 888, 1132, 1165,
1275

Clearing factor, 1143

Clostridium, 1086, 1180, 1931

Clothing, 660

Coe-activating enzyme, 694

Cobalt®, 779

Cobaltoprotein, 486

Cocarboxylase, 1795

Coenzyme A, 221, 698, 1095, 1888

Colchicine, 501, 1295, 1610

Cold, 130, 147, 150, 182, 274, 284, 290, 308,
316, 317, 377, 383, 408, 435, 450, 523,
594, 640

Collagen, 262, 1055

Colloids, 170, 1358

Color vision, 204

Complement, 1695, 1894, 1906, 1968, 198),
1983, 2026

Complement fixation, 1093, 1955, 1964

Conalbumin, 813

Conditioned reflexes, 50

Conditioned response, 292

Conjunctival vessels, 1515

QoQ

eeeoe.2e<seeeen

plume 15

nomethyl,

656, 1204,
1419, 1440,
1508, 1524,

, 389, 395,
1114, 1707,
1849, 1856,

1257, 1536,
, 822, 937,
1374, 1445,
1139

1005, 1038,

1253, 1425,

), 271, 407,

1132, 1165,

1968, 198,

55, 1964

March 1956

Connective tissue, 337, 1705

Constipation, -}588

Contraction, 34, 538

Convulsants, 111, 185

Cooling, 592

Coombs test, 1966

Copper, 1801, 1838

00:2 production, 1101

Coprostanol, 1114

Corn, 1830, 1854

Coronary embolization, 644

Coronary flow, 23, 47, 54, 99, 145, 169,
333, 342, 533

Coronary vasodilators, 1453, 1456, 1630

Corticosteroids, 27, 775, 989

Corticosterone, 743

Cortisol, 743, 1711

Cortisone, 8, 81, 165, 175, 190, 217, 234,
269, 285, 653, 997, 1327, 1362, 1569, 1586,
1644, 1656, 1659, 1691, 1698, 1734, 1750,
1780, 1933, 1944, 1958

Corynebacterium, 1487

CO: tolerance, 105

Coumarin, 1757

Coupled phosphorylation, 968

Coxsackie viruses, 1932

C-reactive protein, 1093

Creatinine nitrogen, 1870

Creatinuria, 1781, 1846, 1886

Croton oil, 1705

Cuneate nucleus, 9

Curare, 476

Cyanide, 859, 1418, 1559

Cyclamate, 1437

Cycloserine, 1344

Cyst, 1673

Cysteamine oxidase, 1129

Cysteic acid, 1039

Cysteine oxidation, 1109

Cystine, 248, 1035

Cytidine, 224

Cytochemistry, 28

Cytochrome, 751, 940, 1226, 1681

Cytochrome oxidase, 959, 1094, 1233

Cytoplasm, 1220

Cytosine, 894, 1082

Daphnia magna, 390

DDT, 1363

DDT-dehydrochlorinase, 984

Decarboxylase, 1198

Decarboxylation, 1994

Decerebration, 638

Dehydration, 239, 326

l-Dehydrocortisol, 1168

|-Dehydrocortisone, 1168

Dehydroepiandrosterone, 310

24-Dehydroergosterol, 1048

Dehydrogenase, 763, 999, 1090, 1148,
1161, 1227, 1231, 1639

Deiodination, 1123

Denaturation, 979, 1030

Denitrifying bacteria, 1226

2-Deoxyglucose, 1254

Deoxypentose nucleic acid, 750

Deoxyribonucleic acid, 162, 882, 1146,
1153

Deoxyribosidic compounds, 1142

Desoxyribonucleic acid, 348, 381, 814,
1342, 1696

Detoxication, 1092

Dextran, 544

SUBJECT INDEX

Diabetes, 37, 132, 164, 183, 519, 1024,
1058, 1308, 1644, 1795, 1845

Diaminopimelic acid, 851

2,6-Diaminopurine, 1271

Diamox, 78, 220, 270, 467, 488

Diaphragm, 1156

6-Diazo-5-oxo-1-norleucine, 774

Dibenzyline, 327, 350, 1292, 1506

1,2-Dibromo-3-chloropropane, 1459

Di-deoxyribonucleotides, 1081

Diet, 33, 255, 379, 389, 428, 603, 718, 742,
1647, 1676, 1687, 1707, 1727, 1730, 1733,
1737, 1740, 1753, 1761, 1769, 1793, 1802,
1803, 1805, 1807, 1819, 1830, 1840, 1849,
1854, 1866, 1867, 1873, 1887

Di-ethionine, 839

Diethylstilbestrol, 678, 1877

Diffusion coefficients, 666

Digestion, 374, 1210, 1772

Digitalis, 1494, 1622

Digitoxin, 1345, 1528, 1533, 1534, 1581,
1617

Digoxin, 1617

Dihydrohydroxymorphinone, 1611

Dihydromorphinone, 1310

Dihydromurexine, 1343

Dihydropyrimidine nucleosides, 864

Dihydroxyfumaric acid, 1127

Dihydroxyphenylethylamine, 972

17a, 21-Dihydroxyprogesterone, 1125

Diiodotyrosine, 1123

Diisopropylfluorophosphatase, 1037

Diisopropyl phosphorofluoridate, 1135

Diketosuccinate, 958

Dilantin, 155, 1312

Dimerization, 931

Dimethylaminoethanol, 1185

Dimethylthetin, 1169

Dimethylthetin-homocysteine methyl-
pherase, 799

Dinitrofluorobenzene, 1253

Dinitrophenol, 120, 754, 1562

Dinitrophenyl-polyvalyl-proteins, 1193

3,3’-Diodothyronine, 1389

Diphenyline, 796

N, N’ - diphenyl - p - phenylenediamine,
1783, 1794

Diphenydramine, 1637

2,3-Diphosphoglycerate, 1217

Diphosphopyridine nucleotide, 752, 935

Diphtheria, 1948, 2033

Disodium adenosine triphosphate, 648

Disulfide bonds, 1960

Diuresis, 177, 260, 351, 540, 1286

Diuretics, 1371, 1476, 1591

Diving, 375

DNA, 692, 945, 1082, 1923, 2010

DOCA, 1558

DOPA, 1155

DPN, 554, 867, 906, 978

DPNH, 675, 699

Dream, 144

Drosophila, 246

Drugs, 140, 1376, 1438, 1580

Dyes, 167, 1718, 1724

Ecuo virus, 1932, 1957
Eclampsia, 1733

Edema, 85, 114, 489

Egg yolk, 717, 1245, 1843
Elastase, 1513, 1752
Electric eel, 536

XXiii

Electrical activity, 366, 376, 567, 1032

Electrocardiogram, 280, 424, 528, 558

Electrochromatography, 927

Electroencephalogram, 114, 219, 510,
560, 584, 635, 1294, 1296, 1355, 1479,
1563, 1600

Electrolytes, 34, 108, 153, 190, 238, 277,
330, 344, 386, 391, 540, 546, 566, 578, 671

Electromyogram, 604

Electron microscope, 1676, 1986

Embryonic development, 1844

Emphysema, 220, 465

Encephalitis, 1905, 1927, 1933

Encephalomyelitis, 938, 1824, 1979

Endocarditis, 7, 1684, 1721

Endocrines, 609, 610, 1672, 1732

Endotoxins, 1890, 2015

Endplate potentials, 77

Enheptin, 1361

Enthanolamine, 1246

Enzymes, 40, 55, 251, 300, 393, 431, 434,
472, 473, 483, 512, 738, 1078, 1103, 1111,
1112, 1148, 1338, 1639, 1646, 1665, 1681,
1693, 1725, 1745, 1752, 1806, 1910, 2009

Eosinopenic response, 1416

Epidemic keratoconjunctivitis, 1937

Epimerase, 902, 907

Epinephrine, 1, 72, 125, 137, 180, 247, 327,
342, 350, 356, 597, 655, 719, 972, 1054,
1405, 1497, 1525, 1540, 1550, 1615, 1621,
1637, 2015

Equine abortion virus, 1651, 1717

Equiperdum, 1327, 1636

Ergosterol, 588

Ergothioneine, 1026

Erythrocytes, 69, 167, 233, 254, 257, 266,
456, 475, 494, 566, 586, 728, 983, 992,
1044, 1119, 1695, 1846, 1897, 1945, 1983,
2006

Erythrocytin, 908

Erythropoiesis, 139, 392

p-Erythrose-4-phosphate, 1179

Escherichia coli, 1053, 1110, 1177, 1230,
1755, 1898

Ester, 1810

Esterase, 56

Estradiol, 893, 966, 977, 1124, 1192, 1638

Estrogens, 686, 1097, 1335, 1662, 1671

Estrone, 1250

Ethanol, 354

Ethionine, 1716, 1832

Ethylene oxide, 1262, 1887

N-ethyl-N-hexahydrobenzyl-8-chloro-
ethylamine, 576

N-ethyl-3-piperidyl diphenyl acetate,
1565

Ethyltrichloramate, 237, 1330

Evans blue, 207

Excitability, 328, 606

Excitation, 17

Excretion, 24, 61, 1880

Exchanges, 344

Exercise, 169, 336, 340, 645, 659

Eye, 49, 632

Fasting, 132, 184, 599

Fat, 184, 193, 458, 553, 708, 1100, 1676,
1692, 1730, 1760, 1762, 1772, 1793

Fatty acids, 97, 187, 194, 215, 408, 573,
588, 850, 1065, 1197, 1234, 1767, 1772

Fern, 135

Ferritin, 19, 1128, 1292

XXIV

FD &C Orange, 1315, 1373

FD & C Red No. 32, 1373

Fibrillation, 109, 267, 424

Fibrinogen, 1270, 1743, 1746

Fibrinolysis, 358, 631, 948

Fibroblasts, 298, 1035, 1926, 1939, 1995

Fish, 649

Flavonones, 724

Flavin peptides, 932

Flavonoids, 1283

Flavoprotein, 706

Fluid, 407, 589

Fluorine, 868, 960, 1014, 1836, 1857

9-a-Fluorohydrocortisone, 1596

Fluoromar, 1624

o-Fluorophenylalanine, 1184

Fluoropyruvate, 812

Fluoropyruvic acid, 744

Folic acid, 840, 1132, 1165, 1740, 1784,
1857, 1868, 1875

Food intake, 1835

Formaldehyde, 942, 1074, 1992

Formalin, 2016

Formamidinoglutaric acid, 1031

Formate, 345, 795, 876, 1087, 1183, 1249

Formiminoglycine, 1086

N'°-formylfolic acid, 1031

a-N-formylglycinamide ribotide, 1240

N'®-formyltetrahydrofolic acid, 916

Forssman antiserums, 2012

Freezing, 223

Frenquel, 1440

Frostbite, 658 ‘

Fructose, 183, 1853, 1878

Fucose, 843

Furfurylideneacetone, 1418

Grantee, 684, 794, 1266

Gall bladder, 1647

Galvanic skin reflex, 638

Gamma globulin, 1703, 1947, 1960, 1983,
2005

Ganglia, 1387, 1501

Ganglionic blockade, 1488 1529, 1549,
1590, 1601

Gas analyzer, 1349

Gastric mucosa, 116, 129

Gastric secretion, 64, 138, 236, 321, 625,
668, 1718

Gas tensions, 283

Gastrointestinal tract, 171, 374

Gecko, 134

Gelatin, 1042

Gelatinase, 1055

Genetics 396

Globulins, 1145

Glomerulonephritis, 1684, 1703

Glucagon, 202, 519, 1182, 1738

Glucogenesis, 1160

Gluconokinase, 970

3-0-a-D-glucopyranosyl-D-glucose, 1063

Glucose, 163, 164, 183, 285, 637, 691, 1004,
1058, 1096, 1208, 1238, 1254, 1261, 1815,
1878, 1909

d-Glucosamine, 438, 1113

Glucose-1-phosphate transphosphoryl-
ase, 1162

Glycine, 1885

N-glucosylglycine, 1110

Glucuronidase, 147, 819

Glucuronolactone, 806, 1323

Glutamate, 697, 968

FEDERATION PROCEEDINGS

Glutamic acid, 741, 869, 896, 1039, 1127,
1263

Glutamine, 711, 740, 863, 1126, 1594, 1909

y-Glutamyl] phosphate, 976

Glutethimide, 1352

Glyceric acid, 721

Glycerol, 928, 947, 1809

Glyceryl methylthiol ester, 724.

Glycinamide ribotide, 857, 878

Glycine, 778, 801, 834, 842, 905, 1869

Glycocyamine, 1067

Glycogen, 32, 163, 202, 471, 885, 898, 1190

Glycogenesis, 988

Glycogen synthesis, 758, 1156

Glycolic acid, 1213

Glycolysis, 824, 845, 1044, 1156

Glycoprotein, 938, 2027

Glycosides, 1452

Glycyrrhiza, 362

Goat, 11, 12

Gold, 485, 1719

Goldthioglucose, 1835

Gonadectomy, 760

Gonadotrophin, 29, 423

Gonads, 423, 458, 512, 608

Growth, 95, 135, 173, 324, 346, 380, 455,
626, 663, 909, 1099, 1717, 1791

Guanine, 711, 1038

Guanosine-5’-phosphate, 1038

Guanosine triphosphate, 982

Guatemala, 1868

Hatching stimulant, 1107

Headache, 1515

Hearing, 313, 353, 489, 510, 627, 629

Heart, 15, 24, 34, 57, 58, 59, 71, 85, 101,
159, 178, 241, 243, 267, 296, 302, 330, 350,
371, 418, 451, 470, 498, 513, 528, 529,
532, 577, 623, 649, 879, 1163, 1188, 1257,
1346, 1351, 1385, 1660, 1689, 1700, 1721,
1753, 1942

Heat, 130, 334, 372, 411

HeLa cells, 696, 855, 1756, 1891, 1909,
1919, 1920, 1961, 1962, 1998

Helenine, 1903

Heliotrine, 1545

Heme synthesis, 1144

Hemoconcentration, 435

Hemocyanin, 1950

Hemodialysis, 1701

Hemodynamics, 52, 86, 229, 243, 306,
384, 443, 469, 481, 503, 543, 571, 577,
581, 591, 595, 602, 642

Hemoglobin 25, 364, 505, 764, 1041, 1754,
1855

Hemoglobinuria, 1833

Hemolysis, 62, 223, 1936

Hemolytic disease, 2007

Hemophilia, 98, 1746

Hemorrhage, 191, 336, 673, 1040

Heparin, 264, 298, 305, 429, 573, 831, 925,
1042, 1062, 1084, 1474, 1743

Hepatic coma, 1587

Hepatitis, 1651, 1717

Heptacyclazine, 1396

Hepatoma, 1142

Heptuloses, 1279

Hesperidin methyl] chalcone, 1557

Hexamethyloeneimines, 1378

Hexokinase, 1025, 1136

Hexosamine, 1507

Hibernation, 301, 373, 492

Volume 1§

Histamine, 4, 399, 1137, 1414, 1427, 1517,
1720

Histidine, 682

Histones, 771

Homocysteine, 1169

Homogentisic acid, 1277

Homoprotocatechuic acid, 1155

Homovanillic acid, 1155

Hufnagel valve, 586, 1721

Hyaluronic acid, 735, 788

Hydantoin, 1325, 1521

Hydration, 1049

Hydrazine, 1493, 1512

Hydrocortisone, 549, 617, 811, 1675, 1839

Hydrogen, 509, 978, 1161

Hydrogen ion concentration, 499, 590, 654

Hydrogenase, 954, 1011, 1064

Hydrolysis, 1810

118-Hydroxy-Atandrostene-3-17-dione-
4-C¥4, 727

19-Hydroxyandrostenedione, 802

3-Hydroxyanthranilic acid, 872, 1024

Hydroxy apatite, 1195

Hydroxyaspartic acid, 1127

8-Hydroxybutyric acid, 1231

17-Hydroxycorticosteroid, 413, 490

14-Hydroxydihydromorphinone, 1310

8-Hydroxyisobutyric dehydrogenase,
1108

Hydroxylamine reductase, 1174

Hydroxylase, 1009, 1125

Hydroxylation, 723, 957, 1134, 1200

8-Hydroxy-8-methylglutaric acid, 1121,
1248

8-Hydroxy-7-methylguanine, 1247

5-Hydroxymethyluracil, 818

N(4-Hydroxy-1-naphthyl)-isomalei-
mide, 1077

6-Hydroxynicotinic acid, 875

p-Hydroxyphenyllactic acid, 849

p-Hydroxyphenylpyruvic acid, 1071,
1277

21-Hydroxypregnanedione, 1564

17a-Hydroxyprogesterone, 1125

Hydroxyproline, 677, 883, 1068

1-3-Hydroxy-N-propargyl morphinaa,
1495

118-Hydroxytestosterone, 1134

5-Hydroxytryptophan, 1311, 1605

3-Hydroxytyramine, 1367

Hypercapnia, 78

Hypercholesteremia, 1858

Hyperheparinemia, 1084

Hyperkalemia, 451

Hyperlipemia, 128, 157

Hyperpnea, 290

Hypertension, 133, 208, 210, 234, 286, 307,
309, 325, 370, 409, 413, 889, 1455, 1557,
1595, 1648, 1666, 1685, 1708, 1729

Hyperthermia, 3, 181, 585

Hyperthyroidism, 470

Hyperventilation, 179, 643

Hypervitaminosis A, 1783

Hypnotic drugs, 1468

Hypoglycemia, 334

Hypokalemia, 109

Hypoproteinemia, 128

Hypotension, 76, 333, 1726

Hypotensives, 1500, 1503, 1526, 1552, 163)

Hypothalamus, 12, 117, 268, 403, 474,
527, 561

Hypothermia, 27, 54, 86, 104, 453, 54,
1319, 1398, 1410, 1430, 1499, 1502, 1570,
1616, 1670

Ket
i-Ket

«Ket
«Ket
«Ket
11-Ket

17,

1715,
Kineti

plume 15

1427, 1517,

1675, 1839

99, 590, 654
7-dione-

2, 1024

, 490
1e, 1310
Jrogenase,

1200
cid, 1121,

1247

malei-

t, 286, 307,
455, 1551,
29

1552, 168
403, 47,

453, 545,
502, 1570,

March 1956

Hypoxanthine, 710

Hypoxemia, 371°

Hypoxia, 5, 25, 112, 195, 214, 437, 495,
§22, 523, 622, 777, 1648, 1694

Leidasoleglycerol phosphate, 682

#@-Iminodiproprionitrile, 17

Immobilization, 803

Immunization, 1049, 1900, 1916, 1985,
1956, 1959, 1966

Indoles, 281

Infections, 1673, 1949

Inflammation, 1659

Influenza virus, 923, 1464, 1475, 1907,
1928, 1930, 1934, 1938, 1944, 1986

Inhibition, 1085, 1988

Inosine hydrolase, 946

Inosinic acid, 982

Inositol, 415

Insecticides, 1380

Insulin, 39, 166, 203, 261, 285, 516, 556,
637, 701, 758, 815, 930, 936, 1024, 1058,
1152, 1156, 1254, 1679, 1683, 1738, 1982

Intestine, 158, 398, 541, 585, 1561, 1677,
1751

Intrinsic factor, 808, 1589

Inulin, 124

Iodine, 226

Iodine; I'3!, 962, 1671

Ions, 74, 90, 764, 1428, 1444, 1461, 1516,
1608, 1613

loresin, 962

Iproniazid, 1334

Iron, 756, 777, 1045, 1064, 1128, 1299, 1706,
1801, 1883

Isoagglutinins, 1908, 2007

lscitric dehydrogenases, 1163

Isohemagglutinins, 2029

Isoleucine, 730, 1222, 1770

Isomerase, 1203

Isoniazid, 1079, 1481

Isoproterenol, 1726

Itaconate, 712

Hiienese virus, 1962, 2001, 2025
Jejunum, 245, 357

Keto acids, 763

«Keto acid keto-enol tautomerase, 1071

§-Ketoadipic acid, 929

-Keto-3-deoxy-7-phospho-D-gluco-
heptonic acid, 1179

§-Ketodipyl CoA, 929

1-Ketoestradiol-178-16-C™, 977

Ketogenesis, 605

aKetoglutarate, 618, 896

«Ketolic steroids, 1187

«Ketols, 269

Ketones, 37

1i-Ketosteroid, 341, 410

Kidney, 36, 61, 118, 124, 147, 151, 153, 157,
177, 206, 217, 230, 234, 240, 252, 256, 260,
M0, 272, 284, 291, 297, 309, 362, 370, 385,
304, 405, 450, 452, 480, 499, 531, 567, 578,
596, 607, 620, 646, 651, 654, 755, 1040,
1182, 1899, 1641, 1645, 1648, 1665, 1668,
1669, 1671, 1675, 1684, 1701, 1703, 1708,
1715, 1745, 1753, 1841

Kinetics, 431, 999

Krebs cycle, 449, 930

Powe ing

SUBJECT INDEX

Kwashiorkor, 1872
Kynurenine, 1079
Kynurenine transaminase, 1010

e-Lactalbumin, 1280

Lactaminic acid, 886

Lactate, 164, 311

Lactic acid, 112, 622, 1167

Lactobacillic acid, 1003

Lactoperoxidase, 1036

Lactose, 1266

Lanosterol, 1056

Lead poisoning, 326

Learning, 117, 143, 436, 461

Leprosy, 1943

Leucocytes, 164, 406, 1119, 1249

Leucyl-AMP, 785

Leukemia, 683, 1680, 1728, 1740, 1742, 1780

Levarterenol, 1621

Lidocaine, 846, 1400

Life span, 69

Light, 135, 1682

Linoleic acid, 1884

Linolenic acid, 1023

Lipase, 158

Lipemia, 305, 1291

Lipids, 165, 193, 273, 616, 1664, 1683, 1685,
1692

Lipogenesis, 703, 1034, 1228, 1679

8-Lipoglobulin, 716

Lipopolysaccharides, 1949

Lipoproteins, 365, 385, 853, 984, 1143,
1212, 1245

Liver, 18, 19, 32, 39, 94, 120, 147, 160, 194,
202, 215, 217, 235, 239, 249, 263, 320, 363,
383, 398, 459, 460, 483, 486, 554, 610,
691, 767, 918, 988, 1034, 1128, 1225,
1287, 1651, 1679, 1687, 1688, 1698, 1736,
1737, 1738, 1757, 1785, 1795, 1837, 1849,
1852, 1866, 1904, 1991

Localization, 113

Locomotion, 140

Luciferase, 127, 1017

Luciferin, 1017

Lungs, 38, 73, 75, 79, 115, 141, 199, 286,
430, 437, 478, 557, 615, 1678

Lupus erythematosus, 1943

Lymph, 596, 1650, 1719

Lymph nodes, 1929

Lymphaties, 222, 250

Lysergic acid, 46, 560, 565, 1395, 1409, 1440

Lysine, 248, 1147, 1194, 1768, 1777, 1817,
1822, 1871

Micceogichalina, 542
Macromolecules, 42, 250
Magnesium, 471, 492, 1043, 1491, 2025
Malic dehydrogenase, 1259, 1265
Mammary tissue, 605

Marine bacteria, 998

Mast cells, 831, 1137, 1646, 1663
Mastitis, 1732

Measles, 1990

3’ MeDAB, 740

Menadione, 809

Menstruation, 85

Meperidine, 1466
Mephentermine, 198
Meprobamate, 1305, 1419
Mercaptalbumin, 931
6-Mercaptopurine, 1478

XXV

Mercurials, 351, 639, 1371, 1476, 1591

Mescaline, 1432, 1467, 1592

Mesentery, 1663

Metabolism, 8, 13, 18, 53, 81, 88, 96, 108,
151, 194, 198, 228, 237, 240, 241, 299, 312,
354, 382, 394, 422, 426, 438, 473, 1421,
1675, 1737, 1740, 1773, 1793, 1796, 1800,
1827, 1919

Metals, 1211

Metamorphosis, 833

Metacholine, 1509

Methemoglobin, 1559

Methionine, 646, 756, 791, 1022, 1035,
1185, 1825, 1832, 1861

A-methopterin, 1249

Methoxychlor, 1618

P-methoxyphenylalanine, 772

P-methoxyphenylpyruvate, 772

3-Methylaminoisocamphane hydrochlo-
ride, 668

Methylcellulose, 1548

6-Methyl-A‘-desoxymorphine, 564

3’-Methy1-4-dimethylaminoazobenzene,
759, 1172

Methyl histidine, 770

Methylmalonyl CoA, 821

w-Methylpantothenate, 989

5-Methyl-5-phenylhydantoin, 1505

Methyl-a-phenyl-2-piperideneacetate,
1522

Methyl synthesis, 1087

Methy] testosterone, 839

5-Methyltryptophan, 702

Metrazol, 225

Micrococcus, 40

Microsomes, 934, 986, 1018

Milk, 630, 1014

Millet, 1830

Minerals, 379, 455, 1195

Mink, 1178, 1825

Mitochondria, 174, 260, 906, 1001, 1018,
1052, 1117, 1164, 1196, 1259, 1681

Mitochrome, 1072

Mitoses, 107, 501

Mitral stenosis, 243

Molecular weight, 1190

Molybdenum, 895, 1064, 1838

Monomerization, 1952

Morgan-Elson reaction, 919

Morphine, 1310, 1384, 1391, 1397, 1433,
1434, 1447, 1619, 1633

Mortality, 65, 1649

Motility, 1657

Motion sickness, 1538

Movement recorder, 1284

Mucopolysaccharides, 1106, 1291

Mucormycosis, 1644

Multiple myeloma, 1083

Multiple sclerosis, 1067

Muscle, 13, 67, 68, 90, 101, 149, 176, 197, 279,
282, 296, 302, 340, 386, 391, 393, 462, 517,
524, 525, 538, 614, 621, 648, 1470, 1652,
1660, 1681

Mutarotase, 936

Mutation, 1910

Myasthenia gravis, 440

Mycrobacteria, 1998

Myleran, 1489

Myocardial infarction, 280, 1689

Myofibrils, 1652

Myokinase, 1050

Myosin, 63, 360, 848, 874, 892, 1188

Myrothecium verrucaria, 897

Myxomyosin, 1220

XXxvi

Natorphine, 1434, 1439, 1447, 1633

Nasal ‘epithelial’ cell, 1939

Necrosis, 1731

Necturus, 230

Nematode, 1107

Nephritis, 1313, 1715

Nephrosis, 1006

Nephrotoxic serum, 1300

Netropsin, 1636

Nerve, 96, 121, 182, 315, 335, 466, 468,
477, 482, 498, 535, 539, 546, 559, 612, 635,
661

Neuromuscular blockade,
1463, 1529, 1535, 1626, 1629

Neurospora, 1022, 1598, 2009

Newcastle disease, 2025

Niacin, 1774, 1799, 1812, 1827, 1854

Nicarbazin, 1350

Nickel carbonyl, 1209

Nicotine, 533, 689, 1590, 1615

Nicotinic acid, 872, 875

Nietroesterones, 1250

Nikethamide, 1547

Nitrate, 297, 525

Nitrite reductase, 1174

Nitrofurantoin, 1306

Nitrogen, 255, 741, 1761, 1764, 1769, 1774,
1829, 1843, 1870, 1889

Nitrogen mustard, 249

Nitrous oxide, 1563

Nodes of Ranvier, 535

Nonprotein sulfhydryl, 1098

Norepinephrine, 1, 72, 180, 327, 342, 350,
356, 1054, 1525, 1555, 1615, 1634

Nose, 209

Nuclease, 2010

Nuclei, 552, 1687

Nucleic acid, 710, 795, 876, 1252, 1724,
1755

Nucleohistones, 771

Nucleoproteins, 263, 1066

Nucleosides, 946, 983, 992, 1243, 1309

Nucleotides, 901, 1053, 1153, 1249, 1309

Nutrition, 1667, 1692, 1821

Nystatin, 963

1374, 1392,

Oxgston hypothesis, 928

Olfaction, 43, 91, 613

38-ol-steroid dehydrogenase, 1766

Omega-methyl-pantothenate, 1677

Opsin, 1235

Optical isomers, 1612

Ormosia panamensis, 1503

Ornithine, 837

Orosomucoid, 555

Osmoreceptor, 113

Osteoarthritis, 1727

Osteoporosis, 1661

Ouabain, 1608, 1632

Oudin serum agar technique, 1974

Ovaries, 367, 423, 444, 667

Ovariectomy, 1827

Ovulation, 189

Ovum, 8

Oxalic acid, 917

Oxalo-acetate, 618

Oxaloglycolate, 958

11-8-oxidase, 1200

Oxidation, 26

Oxidative phosphorylation, 726, 765, 805,
845, 974, 1051, 1199, 1293, 1562, 1583,
1585, 1602

FEDERATION PROCEEDINGS

Oximeter, 465

Oxygen consumption, 5, 71, 73, 88, 99,
342, 449, 462, 640, 652

Oxygen toxicity, 72, 404, 445

Oxygen, 25, 35, 216, 231, 311, 343, 349, 364,
464, 468, 1009

Oxyhemoglobin, 509

Oxyluciferin, 1017

Oxytocin vasopressin EEG, 1497

Pain, 1301

Palmitic acid, 762, 1771

Pancreas, 173, 402, 602, 1693, 1752, 1834,
1846

Pancreozymin, 789

Pantoate, 995

Pantothenate, 995

Pantothenic acid, 989, 1765, 1787, 1858,
1860, 1888

Papain, 939, 1214

Papaverine, 1577

Paper chromatography, 1264, 1420

Paper electrophoresis, 236, 505, 1797

Parabiosis, 1639

Paraldehyde, 1451

Parathyroid, 206

Parkinson disease, 1369

Parotid, 20

Pellagra, 1830

Penicillamine, 687, 1285, 1820

Penicillin, 1191, 1868

Pentamethylenetetrazol, 1329

Pentobarbital, 1303, 1314, 1523, 1539, 1600

Pentose, 943, 1152

Pentosuria, 1886

Pepsin, 1157, 1260

Peptic ulcer, 547, 1546

Peptidase, 601, 707

Peptides, 1193

Pertussis vaccine, 1890, 1978

Phagocytes, 1712

Phagocytosis, 253

Phenobarbital, 1521

Phenols, 784

Phenylketonuria, 634

Pheonthiazines, 1302

Phlorizin, 394

Phosphatases, 400, 693, 1115, 1518, 1606

Phosphates, 46, 138, 233, 254, 517, 607,
1044

Phosphatides, 669, 1797

Phosphodiesterase, 1081

Phosphoenolpyruvate, 1179

Phosphoglyceride, 1080

Phospholipids, 899, 1005, 1020, 1246, 1264,
1614

Phosphopeptides, 1135

5-Phosphoribosylamine, 857, 878

Phosphorus, 53, 92, 796

Phosphorylase, 841, 956, 1091

Phosphorylation, 260, 405, 765, 913, 1013,
1072, 1076, 1170

Photophosphorylation, 847

Photosynthesis, 1214

Phototropism, 502

Phytic acid, 1149

Picolinic acid, 1024

Pilocarpine, 534

Piperazines, 178

Pitressin, 30

Pituitary, 20, 137, 189, 205, 228, 252, 346,
359, 602, 637, 1662, 1672, 1722, 1727, 1765

Volume Bar

Placenta, 518, 901

Plague, 1893 (Qe

Plant juices, 53 fev

Plasma, 11, 187, 192, 248, 775, 786, 109, 9”
1139, 1616, 1642, 1746, 1924 Quine

Plasmapheresis, 2002

Plasmin, 1290, 1975

Plasminogen, 619, 1297

Platelets, 51, 92, 126, 401, 949

Pneumothorax, 479, 557

Polarography, 109, 434, 550

Poliomyelitis, 1653, 1891, 1900, 1903, 1932,
1962, 1967, 1969, 1992

Poliomyelitis virus, 798, 825, 927, 142,
1669, 1895, 1909, 1919, 1955, 1964, 1995,
1993, 2013, 2016, 2034

Polydipsia, 12

Polyenes, 1159

Polyglutamates, 170

Polyneuritis, 2023

Polynucleotides, 734, 891, 1239

Polypeptides, 1181

Poiysaccharides, 631, 1656, 1690

Polyvinylpyrrolidone, 1963

Porphyrins, 967

Posture, 484

Potassium, 2, 121, 238, 282, 319, 386, 3%,
427, 451, 452, 475, 549, 725, 758, 998,

1287, 1665, 1689, 1691, 1798, 1841
Potentials, 293 1603
Precipitin reaction, 1984 Respit
Prednisolone, 1479 315,
Prednisone, 1137, 1479 448,
Pregnancy, 85, 122, 323, 332, 376, 460, 504,
497, 522, 564, 844 Resus
Prephenic acid, 849 Retin
Pressor amines, 1332, 1394, 1412 485,
Procaine, 1353 Retine
Prodigiosin, 1131 Tham
Progesterone, 29, 994 Rheur
Prolactin, 1095 hizo)
Properdin, 1913, 1949, 1965, 2025 Rhodc
8-Propiolactone, 1682 Rhode
Propionate, 821, 969, 1074, 1180 Ribofl
Propylthiouracil, 1958 tibon
Protamine, 287, 827 tibon
Proteases, 1278 1075,
Proteins, 10, 211, 345, 454, 457, 496, 568, i tibon
630, 636, 834, 880, 1047, 1083, 1141, 1187, Ribose
1184, 1243, 1645, 1672, 1693, 1708, 179, ff Mbosi
1730, 1738, 1788, 1792, 1793, 1855, 1868, f§ Hibs
1874, 1879, 1970, 2018 mp
Proteinuria, 1006 paket
Proteolipids, 938 a
Proteolysis, 1699
Prothrombin, 278, 964, 1151, 1642, 1702
Protoporphyrin, 674 Sui
Pteroylglutamic acids, 888, 1275, 184 7
Pulse wave, 503 “td
Purine, 382, 941, 1206, 1240, 1309, 1755, tupon’
1971 win,
Putrescine, 1201 iatura
Pyridine nucleotides, 809, 915, 937, 991, H Sintil
999, 1224, 1774 Slero}
Pyridoxal, 1104, 1144 Scorbu
Pyridoxine, 793, 1709, 1781 Sero-s1
Pyrimethamine, 1578 Seurv;

Pyrimidines, 860, 941 §DA,

Pyrogens, 804, 1473, 1530 Seoobe
Pyrophosphatases, 1150 tee
Pyrophosphate, 934, 1017 eg
Pyruvate, 96, 101, 311, 1177 Selenir

‘olume 16 Byarch 1956

5, 786, 100,

, 1903, 1932,

, 927, 14%,
1964, 1985,

9, 386, 3%,
, 758, 998,

341

, 376, 460,

, 496, 555,
1141, 118),
(708, 1720,
1855, 1863,

42, 1702
5, 1844

309, 175%,

937, 991,

Quenzyme, 841 *
(fever, 2014

Quantum efficiency, 739
quinolinic acid, 872, 890

Radiation, 65, 221, 244, 289, 406, 441,
491, 493, 628, 742, 1299, 1699, 1751
Radioactive metabolites, 1775
Radiobiology, 1339, 1340, 1360, 1366, 1411,
1611, 1518, 1531, 1556, 1584, 1606
Radiocolloid, 1317

fadiostrontium, 570, 1328

fauwolfia alkaloids, 1307

Reabsorption, 230, 499

Rectal polyps, 1696

Reflexes, 16, 168, 258, 265, 271, 272, 328,
433, 466, 513, 514, 574, 580, 592, 604, 647,

655

felaxin, 367, 792

Renal pharmacology, 1436, 1446, 1450,
1536

Renin, 208

Reproduction, 1658, 1674

Reptiles, 786

Reserpine, 35, 1334, 1337, 1348, 1356, 1388,
1415, 1419, 1423, 1431, 1440, 1454, 1462,
1465, 1467, 1498, 1508, 1522, 1573, 1592,
1603, 1658

Respiration, 110, 115, 123, 200, 220, 314,
315, 336, 347, 368, 373, 414, 430, 447,
48, 453, 464, 465, 478, 484, 495, 503,
504, 545, 600, 665, 1677, 1756, 1981
Resuscitation, 100

Reticuloendothelium, 156, 288, 292,
485, 522, 673

Retinene, 904

Rhamnose, 843

Rheumatoid arthritis, 811

Rhizopus MX, 1004

Rhodopsin, 904, 1235

Rhodospirillum rubrum, 847, 1170
Riboflavin, 738, 865, 1390, 1829
Ribonuclease, 685, 732, 789, 1117, 2010
Ribonucleic acid, 776, 858, 918, 1060,
1075, 1141, 1230, 1736

Ribonucleoprotein particles, 986
Ribose-5-phosphate, 790

Riboside, 695

Riboside hydrolase, 1202

+Ribulose 5-phosphate, 907

Rickettsiae, 1899, 1995

Ring A, 1111

Ryanodine, 1385, 1426, 1527

ulicylic acid, 1981
filivary gland, 534
sponins, 1627

Sarin, 331, 1030, 1135
futuration, 1223
kintillation, 720, 1219
Sleroprotein, 337
Scorbutic guinea pigs, 945
Sro-survey, 1996
Scurvy, 743, 1711

SDA, 1788

Secobarbital, 1460, 1600
Secretion, 212, 304
Secretin, 1509
Sedimentation, 666
Selenium, 1015, 1417

XUM

SUBJECT INDEX

Semen, 1258
Senecioic acid, 1248
Senna, 1588
Sensitivity, 2018
Septal defect, 371
Sequence studies, 939
Serine, 687, 778, 791, 893, 905, 916, 942,
947, 1033, 1885
Serotonin, 1334, 1348, 1356, 1359, 1393,
1415, 1465, 1467, 1480, 1573, 1605
Serum, 779, 788, 839, 1863
Serum protein, 683, 708, 709, 920, 979,
1015, 1116, 1138, 1237, 1255, 1716, 1750,
1831
Serylhistidylleucylvalylglutamie acid,
1028
Sex hormones, 1827
Sexual differentiation, 990
Shikimic acid, 1179
Shivering, 60
Shock, 175, 177, 274, 398, 420, 543, 563,
575, 576, 948, 1196, 1292, 1452, 1532,
1549, 1554, 1634
Shwartzman reaction, 2015
Sialic acid, 886
Sialoadenectomy, 1241
Sickle-cell, 1041
Sicklemia, 2
Silica, 1659
Silico-molybdate, 913
Silicosis, 1678
Silkworms, 1269
8-Sitosterol, 704
SKF 525 A, 1433
Skin, 85, 172, 226, 259, 289, 312, 491, 500,
658, 829, 1484, 1537, 1720, 1739, 1749
Sleep, 144
Smallpox, 1935
Snake venoms, 154
Sodium, 2, 22, 24, 70, 116, 148, 238, 319,
386, 475, 549, 621, 725, 998, 1437, 1539,
1691, 1790
Somatotrophin, 102, 122, 228, 325, 359,
~ 701, 842, 1357, 1553, 1649, 1765, 1779
Soybeans, 1157, 1861
Soy sterols, 1856
Sparteine, 1383
Spermidine, 1201
Spermine, 1201, 1242, 1595
Sphingolipides, 641
Spinal fluid, 283
Spleen, 174, 1728
Squalene, 395
Staphylokinase, 1918
Starch, 1802
Starfish, 502
Stearic acid, 762
Stellate ganglion, 506
Sterilization, 1682
Steroid, 106, 175, 380, 518, 611, 722, 766,
836, 1111, 1125, 1251, 1764
Sterol, 829, 1175, 1707, 2022
Stibophen, 1320
Stilbestrol, 1221
St. Louis encephalitis virus, 1901
Stomach, 148, 212, 304, 321, 400, 582, 1718
Strandin, 826, 1089
Streptococcus, 1902, 1942, 1988
Streptogenin, 1028
Streptokinase, 1297
Streptolysin O, 1989
Streptomyces griseus, 1238
Stress, 161, 166, 206, 377, 439, 561, 1270,
1793

XXVIli

Stretch receptors, 1579

Striopallidium, 572

Strontium, 1704, 1881

Strychnine, 476

Succinate, 1007

Succinic dehydrogenase, 699, 932, 1011,
1094, 1232

Succinoxidase, 897, 1879

6-Succinylaminopurine, 748

Sucrose, 1213, 1802

Sudan black B, 715

Sugar-amino acids, 1045

Sulfaethylthiadiazole, 1375

Sulfaguanidine, 1758

Sulfatase, 1062

Sulfate, 784, 1105, 1838

Sulfathiazole, 911, 1755

Sulfhydryl, 709, 1041, 1077, 1090

Sulfide, 700

Sulfonamides, 1544

Sulfonated lignin, 1474

Sulfur, 1823, 1857, 1888

Surface tension, 79

Sweat, 196

Sympathectomy, 125

Synapses, 281, 606

Synovial fluid, 1047, 1070

Synthesis, 1032

Syphilis, 1943

Teben poisoning, 1318

TAMe assay, 1642

Tapazole, 623

Tartaric acid, 693, 953, 958

Taurine, 996

Tea, 1836

Temperature, 136, 172, 210, 284, 289, 301
303, 320, 326, 559, 587, 590, 597, 614, 2032

Temperature regulation, 218, 223, 308,
316, 329, 372, 425, 428

Teratogenic action, 774

Testes, 415

Testosterone, 457, 608, 834, 1134

Tetanus toxoid, 2000

Tetrahymena, 805, 898

Tetramethylammonium chloride, 1025

Tetrazolium, 912

Thai, 1807

Thalamus, 265

Theobromine, 768

Theophylline, 768

Thermal inactivation, 2034

Thetins, 1140

Thiamin deficiency, 728

Thiamylal, 1514

4-Thiazolidinecarboxylic acid, 859, 997

Thiobarbital, 1324

Thiocholesterol, 757

Thioctic acid, 1587

Thio-esters of acylated glycine, 1625

Thioketone, 675

Thiopental, 1092, 1282, 1333, 1514

Thiosulfate, 700

Thiouracil, 1582, 1790

Thiourea pleural effusion, 997

Thirst, 372

Threonine, 926, 1222

Thrombin, 922, 1151

Thromboplastin, 251, 1158

Thymidine triphosphate, 950

Thymine, 817, 828, 1276

Thymus, 319, 1485, 1575, 1586, 1710

Thyroglobulin, 1999

Thyroid, 1, 137, 317, 318, 396, 444, 454,
1001, 1241, 1572, 1585, 1671, 1697, 1732,
1758, 1786, 1850, 1852, 1987

Thyrotropic hormone, 80, 205, 761, 1069

Thyroxine, 26, 55, 240, 497, 539, 597, 962,
1265, 1890, 1471

Tidal volume, 123

Tissue, 344, 345, 1643, 1645, 1713, 1793

Tissue culture, 1652, 1899, 1915, 1961,
1993, 2011, 2028

Tissue gas tensions, 624

Tissue slices, 81

Tobacco mosaic virus, 927, 1120, 1186

Tocopheryl, 1013, 1846, 1886

p-Tolyl azo fibrinogen, 820

Tomatoes, 1159

Toxicity, 237, 1791

Toxins, 77, 397, 1893, 1898

Toxoplasma, 1673, 1913

Tracers, 1645, 1664, 1671, 1690, 1740

Transacetalation, 1068

Transamidinase, 837, 1236

Transamination, 921

Transferase, 819, 892

Transformylation, 1240

Transfusion, 98

Transketolase, 728, 902

Trans-L-epoxysuccinate, 1008

Transmethylation, 1169

Transplantation, 1655, 1680, 1686, 1727,
1734, 1778, 1780

Trauma, 549, 863, 1977

Trehalose, 1269

Tremor, 656

Treponema pallidum, 1657

Tricalcium phosphate, 1273

Tricarboxylic acid, 696, 1795, 1882

2,2, 2-trichloro-1-hydroxy ethylcarbamic
acid, 237

Triiodothyronine, 318, 823, 1123, 1958

Trimethadione, 1597

Triose phosphate isomerase, 1103

Tripalmitin, 1100

Tropics, 1883

Tris, 952

Triton, 1143

Trycyclene, 1326

FEDERATION PROCEEDINGS

Trypsin, 705, 1030, 1135, 1154, 1210, 1244,
1278, 1425

Tryptophan, 890, 1078, 1079, 1774, 1799,
1812, 1828, 2009

Tubercle bacilli, 1998, 2028

Tuberculosis, 1958, 1973

Tubule, 725

Tumors, 324, 348, 426, 583, 589, 670, 703,
824, 1016, 1101, 1176, 1267, 1316, 1543,
1655, 1662, 1680, 1694, 1696, 1713, 1714,
1728, 1744, 1748, 1811

Tungsten, 895

Tween, 20

Two-strand structure, 1146

Typhoid fever, 1916, 2004

Typhus, 2014

Tyrosinase, 731, 877, 1822

Tyrosine, 363, 849

UWaessene, 295

Ultraviolet light, 20, 1239, 1643, 1682,
2013

8, y-Unsaturated 3-ketosteroids, 1203

Uracil, 94, 745, 871, 884, 894

Urea, 531, 1030, 1791, 1997

Uremia, 1997

Urethane, 805

Uric acid synthesis, 1033

Uridine, 224, 852, 1274

Urine, 397, 611, 836, 1102, 1165, 1473

Uterus, 360, 376, 496, 626

Vaccines, 1965

Vaccinia virus, 2024

Vagus, 529

Valine, 730, 1191

Vanillin, 975

Vasopressin, 106, 1281, 1558, 1632
Venoconstriction, 521, 2032
Ventilation, 38, 73, 665
Ventricle, 70

Veratridine, 1608

Veratrum, 1341

Vesiculase, 1258

Virus Tz, 1177

Vision, 134, 188, 201, 204, 293, 313, 482

Volu

Visual pigment, 134 i

Vitamin A, 365, 965, 1759, 1783,
1810, 1842, 1864, 1872 E

Vitamin Be, 1813, 1863 Z

Vitamin Bis, 87, 235, 653, 779, 1033,
1654, 1785, 1814, 1847, 1850, 1857, 1%
1873, 1875 4

Vitamin B’s, 1814, 1818, 1887

Vitamin C, 1711, 1839

Vitamin D, 783, 1741, 1779, 1798

Vitamin E, 379, 754, 1781, 1789, 1
1837, 1846, 1866, 1867, 1869, 1882

Vitamin K, 421, 1833

Vocal cords, 200

Vomiting, 66

W ecsrniin antibody, 1976
Water, 153, 171, 190, 320, 428, 456, 603,
Water balance, 532, 671, 749
Weight, 152, 520, 1649

Wilson’s disease, 36

Work, 21

Xanthine oxidase, 1271, 1879

Xanthomatosis, 1831

Xanthosine phosphate, 711, 1038

Xenon, 1520 i

X-irradiation, 15, 66, 76, 82, 95, 162,
238, 249, 266, 276, 419, 420, 445, 500,
562, 598, 599, 749, 795, 862, 1034,
1641, 1648, 1649, 1655, 1663, 1682,
1697, 1699, 1704, 1712, 1723, 1725,
1749, 1750, 1751, 1901, 1921, 1933,
1951, 1959, 2000

Xylitol, 1216

Xylulose, 902, 907, 1216

Yeast, 48, 300, 776, 1051, 1141,
1664
Yttrium, 961, 1116

Zinc, 1492, 2019

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